Methods and composition for diagnosing and treating Pseudoxanthoma elasticum and related conditions

ABSTRACT

Methods and compositions are provided for diagnosing and treating  Pseudoxanthoma elasticum  (PXE) patients and PXE carriers. Methods and compositions are based on the discovery that PXE mutations are located in the MRP6 (ABCC6) gene.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/792,616, filedFeb. 23, 2001, now U.S. Pat. No. 6,780,587 and claims priority to, andthe benefit of U.S. Ser. No. 60/184,269, filed Feb. 23, 2000, the entiredisclosures of which are incorporated by reference herein in theirentireties.

GOVERNMENT SUPPORT

Work described herein was supported in part by Federal Grant No. EY13019. The U.S. Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the field of physiologicaldysfunctions associated with Pseudoxanthoma elasticum. Moreparticularly, the invention is concerned with the identification of agene associated with Pseudoxanthoma elasticum, as well as mutations inthe gene that cause the disease. The present invention also relates tomethods for detecting and diagnosing Pseudoxanthoma elasticum, tomethods for identifying carriers of mutant and normal alleles of thegene associated with Pseudoxanthoma elasticum, to methods for screeningcompounds to identify potential therapeutics for Pseudoxanthomaelasticum, to treatment methods for Pseudoxanthoma elasticum, and touseful cell lines and animal models of the disease.

BACKGROUND OF THE INVENTION

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized bymineralization of elastic fibers in skin, arteries and the retina, thatresult in dermal lesions with associated laxity and loss of elasticity,arterial insufficiency, cardiovascular disease and retinal hemorrhagesleading to macular degeneration.

The skin manifestations are among the most common characteristics ofPXE, but the ocular and cardiovascular symptoms are responsible for themorbidity of the disease. Characteristic skin lesions are generally anearly sign of PXE and were first described by a French dermatologist in1896. Skin lesions are usually detected during childhood or adolescenceand progress slowly and often unpredictably. Therefore, the initialdiagnosis of PXE is sometimes made by a dermatologist. The skin lesionsconsist of yellowish papules and plaques and laxity with loss ofelasticity, and result from an accumulation of abnormal mineralizedelastic fibers in the mid-dermis. Lesions are typically seen on theface, neck, axilla, antecubital fossa, popliteal fossa, groin andperiumbilical areas. A PXE diagnosis can be confirmed by a skin biopsythat shows calcification of fragmented elastic fibers in the mid- andlower dermis.

Another characteristic of PXE is the presence of ocular lesions due tothe accumulation of abnormal elastic fibers in the Bruch's membrane,resulting in angioid streaks. Doyne was the first to describe theseocular streaks in 1889, and Knapp introduced the term “angioid streaks”for their resemblance to blood vessels. The combination of PXE andocular manifestations was initially referred to as theGronblad-Strandberg syndrome, after the names of two ophthalmologistswho independently related the occurrence of angioid streaks to PXE in1929. The majority of PXE patients (approximately 85%) develop ocularmanifestations during their second decade of life. Bilateral angioidstreaks are normally seen as linear gray or dark red lines withirregular serrated edges lying beneath normal retinal blood vessels andrepresent breaks in the Bruch's membrane. The Bruch's membrane is not ina true sense a “membrane” but rather a heterogeneous elastin-rich layerseparating the chorioid from the retina. The elastic laminae of theBruch's membrane is located between two layers of collagen (type I, IIIand IV) which lie in direct contact with the basement membranes of theretinal pigmented epithelium (RPE) and the capillaries in thechoriocapillary layer of the chorioid As a consequence of angioidstreaks, a PXE patient progressively develops a chorioidalneovascularization with a subsequent hemorrhagic detachment of the foveaand later scarring. Optic nerve drusen may also be associated withangioid streaks and results in visual field deficits and even advancedvisual impairment.

Common cardiovascular complications of PXE are due to the presence ofabnormal calcified elastic fibers in the internal elastic lamina ofmedium-sized arteries. The broad spectrum of phenotypes includespremature atherosclerotic changes, intimal fibroplasia causing angina orintermittent claudication or both, early myocardial infarction andhypertension Fibrous thickening of the endocardium and atrioventricularvalves can also result in restrictive cardiomyopathy. Approximately 10%of PXE patients also develop gastrointestinal bleedings and centralnervous system complications (such as stroke and dementia) as aconsequence of systemic arterial wall mineralization. In addition,renovascular hypertension and atrial septal aneurysm can be seen in PXEpatients.

Strikingly, lung abnormalities are not a significant phenotypic featureof PXE, even though pulmonary tissues are rich in elastic fibers.Mineralization of pulmonary elastic fibers has only been noted in a fewpatients.

PXE is usually found as a sporadic disorder but examples of bothautosomal recessive and autosomal dominant forms of PXE have beenreported. Partial manifestations of the PXE phenotype have also beendescribed in presumed carriers in PXE families. Recent reports havelinked both the dominant and recessive forms of PXE to a 5 cM domain onchromosome 16P13.1 However, the mechanisms underlying the physiologicaldefects characteristic of PXE are not understood.

Therefore, there is a need in the art for methods and compositions fordiagnosing and treating PXE and PXE associated phenotypes.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for diagnosing andtreating PXE and PXE associated physiological dysfunctions. According tothe invention, mutations associated with PXE are located in the (MRP6)ABCC6 gene. Therefore, methods for detecting the presence of a mutationassociated with PXE involve interrogating the (MRP6) ABCC6 gene, or aportion thereof, for the presence of one or more mutations that areassociated with PXE. Accordingly, one aspect of the invention providesmethods for identifying individuals that have one or two mutant allelesat the PXE locus. PXE is most often an autosomal recessive disease.Therefore, an individual with two mutant (MRP6) ABCC6 alleles associatedwith PXE will develop symptoms characteristic of the disease. Incontrast, an individual with one mutant (MRP6) ABCC6 allele associatedwith PXE is a carrier of the disease and does not develop full-blownPXE. However, according to one embodiment of the invention, a PXEcarrier may develop mild forms of the characteristic manifestations.Accordingly, a PXE carrier status can be indicative of a predispositionto PXE related symptoms such as eye, skin, or cardiovascular problems.In a preferred embodiment of the invention, genetic counseling isprovided to an individual identified as having a mutation associatedwith PXE in one or both alleles of the PXE ((MRP6) ABCC6) locus.

In another aspect, the invention provides compositions for detecting thepresence of a mutation associated with PXE at the (MRP6) ABCC6 locus. Ina preferred embodiment, an oligonucleotide that hybridizes to the (MRP6)ABCC6 locus is used in a diagnostic assay. In a more preferredembodiment, the oligonucleotide includes a sequence complementary to amutation that is associated with PXE. Alternatively, an antibody-baseddiagnostic assay is used to detect the presence of a mutation associatedwith PXE at the (MRP6) ABCC6 locus.

Other aspects of the invention include therapeutic uses of the (MRP6)ABCC6 gene or protein, drug screening, the identification of (MRP6)ABCC6 homologues in other organisms (including mammalian organisms),cellular and animal models of PXE, the identification of (MRP6) ABCC6functional domains related to the PXE phenotype, the identification ofregulators of (MRP6) ABCC6 expression (mutations in these regulators canalso result in PXE related symptoms), the identification ofgenes/proteins that interact with (MRP6) ABCC6 (alterations in theseinteracting molecules can also cause PXE related symptoms).

Thus, in one series of embodiments the invention provides methods forscreening for the presence of a PXE mutation by interrogating an MRP6nucleic acid obtained from a patient for the presence of a PXE mutation.The screen is positive is the presence of a PXE associated mutation isdetected. A PXE associated mutation is a mutation that causes the PXEphenotype in an individual that is homozygous for the mutation. PXEassociated mutations also causes the PXE phenotype in an individual thatis a compound heterozygote with two different mutant alleles at the MRP6locus, wherein each allele is a PXE associated allele. Nucleic acid isisolated from a patient biological sample, and the biological sample ispreferably blood, saliva, amniotic fluid, or tissue such as a biopsytissue. According to the invention, an MRP6 nucleic acid is a nucleicacid obtained from the MRP6 locus. An MRP6 nucleic acid can be mRNA,genomic DNA or cDNA from the MRP6 locus, or a PCR product of any of theabove. According to the invention, the MRP6 locus includes the MRP6exons, introns, and associated promoter and regulatory sequences in thegenome surrounding the MRP6 exons.

In one series of embodiments, a PXE associated mutation is detected inMRP6 using a nucleic acid based detection assay. Preferred nucleic acidbased detection assays include hybridization assays, primer extensionassays, SSCP, DGGE, RFLP, LCR, DIHPLC, and enzymatic cleavage assays. Inanother series of embodiments, a PXE associated mutation is detected ina protein based detection assay. Preferred protein based detectionassays include ELISA and a Western blot assays. In one embodiment of theinvention, mutation detection assays are provided to screen the MRP6locus or a portion thereof to determine whether a mutation is present.The lack of MRP6 expression or the expression of a physically aberrantform of MRP6 may be sufficient to determine that an individual has a PXEassociated mutation at the MRP6 locus. Alternatively, the determinationthat a mutation is present in the MRP6 locus may not be sufficient todetermine the PXE status of an individual in the absence of informationconcerning the specific identity of the mutation. If such a mutation ispresent, it may be identified according to methods of the invention, forexample by sequencing the region of the MRP6 locus that contains themutation. Once a mutation is identified in a patient sample, the PXEstatus of the patient can be determined according to methods of theinvention. In an alternative embodiment of the invention, specificmutation detection assays are provided to detect a known PXE associatedMRP6 mutation in a patient sample.

In another series of embodiments, the invention provides oligonucleotideprobes or primers and antibodies for use in mutation detection assays orscreens according to the invention.

In another series of embodiments, the invention provides methods forscreening candidate drug compounds to identify therapeutic compounds fortreating PXE patients (individuals that have PXE due to the presence oftwo recessive PXE associated MRP6 alleles, or one apparently dominantPXE allele) or PXE carriers (individuals with one normal MRP6 allele andone allele with a PXE associated mutation).

In another series of embodiments, the invention provides methods fortreating PXE patients or carriers using a normal MNP6 nucleic acid orprotein to restore normal MRP6 function to the individual or to specificcells or tissues or the individual.

In another series of embodiments, the invention provides methods forcreating transgenic or knockout cell lines and animals in order toprovide a model system for PXE.

In another series of embodiments, the invention provides methods foridentifying compounds such as other intracellular proteins that interactwith MRP6 thereby to identify additional therapeutic targets for PXEtreatment.

Accordingly, the invention provides methods and compositions forunambiguously determining the PXE status of an individual. The inventionprovides methods for detecting deletions, substitutions, insertions, andrearrangements in the MRP6 locus that are associated with PXE. Inpreferred embodiments, the invention provides methods for identifyingmutations known to be associated with PXE. Preferred mutations includemutations that affect one or more of the bases in codons 1114, 1138,1141, 1298, 1302, 1303, 1314, 1321 and other codons identified herein asbeing important for normal MRP6 function. Alternatively, the inventionprovides methods to identify mutations that result in non-conservativesubstitutions in the MRP6 locus. In a further embodiment, the inventionprovides assays to detect PXE associated mutations at intron/exon splicesites of the MRP6 gene. The invention also provides methods to detectmutations that affect one or more regulatory elements of the MRP6 gene,including the promoter, the polyA site and other transcriptional ortranslational control sequences.

Methods of the invention are also useful to screen a population in orderto identify individuals with one or more PXE associated MRP6 alleles.According to the invention, these individuals are provided withappropriate genetic counseling in view of their PXE status.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of the MRP6 gene and the surrounding genomicregions. Two (MRP6) ABCC6 mutations that cause PXE are indicated.

FIG. 2 shows the predicted topology of the MRP6 protein and the locationof ten mutations causing PXE.

FIG. 3 shows conserved amino acids in the human MRP6 protein.

FIG. 4 shows co-segregation of the PXE phenotype with the R1141Xmutation in exon 24 of the (MRP6) ABCC6 gene.

FIG. 5 shows segregation of the PPXE phenotype for an apparent autosomaldominant mutation.

FIG. 6 shows a construct for deleting exon 28 in a mouse.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions for diagnosing andtreating PXE and PXE related symptoms. Methods and compositions of theinvention rely in part on the discovery that mutations associated withPXE map to the (MRP6) ABCC6 gene locus on chromosome 16. Accordingly,the invention provides useful PXE related diagnostic and therapeuticmethods and compositions by exploiting wild-type and mutant (MRP6) ABCC6genes and proteins.

I. PXE Associated Mutations in the (MRP6) ABCC6 Gene

a) Mapping of PXE Associated Mutations to the (MRP6) ABCC6 Genetic Locus

Although the first case of PXE was reported by Darier in 1896, most PXEcases have been reported since the 1970s. In most reports, PXE isinherited as an autosomal recessive (AR) phenotype or appears as asporadic phenotype. However, kindreds showing apparent autosomaldominant (AD) inheritance have also been reported. Using DNA frompatients and unaffected family members from 21 unrelated PXE families,the PXE phenotype was linked to the short arm of chromosome 16. A verysignificant linkage with an 8 cM region was demonstrated with a maximumlod score of 8.07. A subsequent haplotype analysis and recombinationmapping reduced the locus from 8 cM to 820 kb where six candidate geneswere identified. The locus was later reduced to less than 600 kb and onecandidate gene was excluded. All 109 exons of the five remainingcandidate genes were screened by a combination of single-strandconformation polymorphism (SSCP), heteroduplex analysis (HA) or directsequencing using genomic DNA from a cohort of 17 unrelated PXE patientsand three unrelated normal individuals. The first six mutations, clearlyassociated with the PXE phenotype, were found in the (MRP6) ABCC6 gene(also known as the ABCC-6 gene). This analysis is described in furtherdetail in Example 2. According to the invention, the MRP6 gene has 31exons as shown in FIG. 1. A 107.7 kb genomic sequence that includes theMRP6 locus is shown in SEQ ID NO: 1. The sequence of SEQ ID NO: 1 showsthe complementary strand of the MRP6 gene. The intron/exon boundariesare as follows (on the complementary strand of SEQ ID NO: 1): Ex1:102783-102748; Ex2: 101180-100998; Ex3: 99296-99171; Ex4: 99031-98903;Ex5: 93798-93676; Ex6: 91594-91533; Ex7: 88207-88076; Ex8: 82954-82757;Ex9: 81524-81347; Ex10: 77528-77367; Ex11: 72268-72176; Ex12:69718-69515; Ex13: 68325-68182; Ex14: 66562-66475; Ex15: 64385-64310;Ex16: 62282-62156; Ex17: 61940-61764; Ex18: 58324-58457; Ex19:56985-56811; Ex20: 55345-55270; Ex21: 52757-52637; Ex22: 49588-49381;Ex23: 45578-45268; Ex24: 42837-42638; Ex25: 41209-41083; Ex26:39226-39125; Ex27: 37453-37307; Ex28: 34674-34516; Ex29: 34437-34271;Ex30: 30412-30218; Ex31: 29881-29773. The mRNA coding sequence for humanMRP6 is shown in SEQ ID NO: 2, and the encoded protein sequence is shownin SEQ ID NO: 3.

b) Identifying PXE Associated Mutations in the (MRP6) ABCC6 Locus

According to methods of the invention, additional PXE associatedmutations were identified in the (MRP6) ABCC6 locus using a combinationof single strand conformation polymorphism (SSCP), heteroduplex analysis(HA) and direct sequencing. Single nucleotide mutations in the (MRP6)ABCC6 gene were identified in several cohorts of individuals originatingfrom the United States, South Africa and several European countries(Belgium, Germany, Holland, Italy and United Kingdom). To confirm thecausative or polymorphic nature of new variants, a control panel of 300alleles (150 normal individuals) was screened and the co-segregation ofthe identified variant and the PXE phenotype was verified. It isnoteworthy that two single-allele mutations (R1141X, R1339C) were foundin control panels of normal individuals indicating that heterozygotemutant (MRP6) ABCC6 alleles can be found in the normal population.However, the missense mutation (R1339C) was identified in thegenetically distinct Afrikaners of South Africa. The frequency ofheterozygote carriers deduced only from the appearance of theseheterozygote mutations is 1.3 percent and is consistent with thecommonly accepted figures of 0.6 to 2.5%. Indeed, while most mutationsappeared to be private, a few have been clearly identified as recurrent(R1141X, R518Q, 3775delT, 16.5 kb deletion between exon 22 and 29. Mostof the mutations (63%) were missense substitutions, 17% were nonsensemutations (5), 13% were frameshift mutations (4 deletions or aninsertions of a single nucleotide) and 7% were likely to affect splicing(2).

Twenty-seven of the mutations (90%) affected the C-terminal half of the(MRP6) ABCC6 protein and particularly the various domains of theC-terminal ATP-binding site, which are encoded by exons 28 to 30, where12 (40%) mutations were clustered. Remarkably, 10 mutations (33%)affected arginyl residues. Eight of these were missense substitutions,suggesting an essential structural or functional role for these arginylresidues in (MRP6) ABCC6.

Large deletions, which are not detected by SSCP or HA, can be identifiedby the loss of heterozygosity of informative polymorphic markers. Sevenhighly informative microsatellites present in a 300 kb regionencompassing both ABCC1 and ABCC6, have been successfully used to detectlarge deletions involving parts or the entire ABCC6 gene. The loss ofheterozygosity can also be efficiently implemented by using severalhighly polymorphic variants present in the ABCC6 gene. The latterapproach was used to detect a partial deletion of the (MRP6) ABCC6 gene,in a compound heterozygous state, in a family with an apparent dominantform of PXE, as discussed in Example 3. A non-limiting list of known PXEassociated mutations at the MRP6 locus are shown in Table 1.

TABLE 1 Known PXE associated mutations at the human MRP6 locus.Mutations Status Effect Nt change Status Origin Exons — 938-939insT ch,ht A  8 R518Q 1553G > A ch, ht a, u 12 F568S 1703T > C ht U 13 L673P2018T > C ch A 16 — 1995delG ch G 16 — 2322delC ht U 18 Y768X 2204C > Ach, ht A 18 — IVS21 + 1G > T ch U Intron 21 R1030X 3088C > T ht A 23R1114P 3341G > C hm Uk 24 S1121W 3362C > G ch G 24 R1138P 3413G > C ch G24 R1138Q 3413G > A ch Uk 24 R1141X 3421C > T all All 24 G1203D 3608G >A 25 — IVS26 − 1G > A ch B Intron 26 W1241C 3723G > C 26 Q1237X 3709C >T ch B 26 — 3775delT ht, hm a, u, h 27 V1298F 3892G > T ht U 28 T1301I3902C > T ch B 28 G1302R 3904G > A ht U 28 A1303P 3907G > C ch B 28R1314W 3940C > T hm U 28 R1314Q 3941G > A ht G 28 G1321S 3961G > A ht U28 R1339C 4015C > T all a, u 28 Q1347H 4041G > C ht U 28 D1361N 4081G >A ch G 29 R1398X 4192C > T ch B 29 I1424T 4271T > C ht U 30 ch =compound heterozygote; ht = heterozygote; hm = homozygote; ivs =intervening sequence

According to methods of the invention, additional PXE associatedmutations can be identified in the (MRP6) ABCC6 locus according tomethods of the invention. For example, single strand conformationpolymorphism (SSCP), heteroduplex analysis (HA), or direct sequenceanalysis can be used to identify additional mutations at the MRP6 locus.In one embodiment, the analysis is performed on genomic DNA.Alternatively, the analysis is performed on cDNA or on exon containingDNA amplification products such as exon containing PCR products.Deletion mutations are preferably detected using diagnostic PCR assaysof genomic DNA and by Southern hybridization according to methods knownin the art. In addition, fluorescent in situ hybridization (FISH)analysis of human chromosome preparations can be used to identify adeletion at the MRP6 locus or a deletion that encompasses all or part ofthe MRP6 locus. Specific mutations are preferably identified using DNAarrays including mutation specific oligonucleotide probes.Alternatively, mutation-specific antibodies can be used to detectmutations that alter an existing epitope or create a new specificepitope on the MRP6 protein. Preferably, specific antibodies are used onproteomic chips to detect protein altering mutations in the MRP6 gene.Mutations can also be detected using mass spectrometry, andmutation-specific mass spectrometer profiles can be generated for MRP6nucleic acid or protein analysis according to methods known in the art.

c) PXE Associated Mutations at the (MRP6) ABCC6 Locus

i) The (MRP6) ABCC6 Gene and Protein

The (MRP6) ABCC6 gene, also known as the ABBC6 gene, encodes anATP-binding cassette transporter (an ABC transporter) belonging tosub-family “C” which includes genes involved in drug-resistance such asMRP1 to 6 (ABCC1-6). (MRP6) ABCC6/ABCC6 encodes a 165 kDa protein thatis located in the plasma membrane and has 17 membrane-spanning helicesgrouped into three transmembrane domains. MRP6 is highly homologous toMRP1 and may act as an efflux pump of amphipathic anion conjugates.Accordingly, in one aspect of the invention, MRP6 transports glutathioneanion conjugates and also anionic drugs. Therefore, an individual thatis a PXE carrier or a PXE homozygote or compound heterozygote may havereduced transport of anionic drugs and may be more receptive tochemotherapy using such drugs. The ABCC family of genes also includesthe cystic fibrosis transmembrane conductance regulator gene (ABCC7 orCFTR) and the sulfonylurea receptor genes (ABCC8 and 9 or SUR).

Therefore, in contrast to genetic changes involved in other elasticfiber diseases such as Supravalvular Aortic Stenosis (SVAS), Marfansyndrome, and Cutis laxa, PXE associated mutations in the (MRP6) ABCC6gene are not directly related to elastic fibers. The (MRP6) ABCC6/ABCC6gene is expressed at relatively high levels in a limited range oftissues, notably in kidney and liver. However, low levels of expressionare also observed in smooth muscle cells and macrophages. According tothe invention, this tissue distribution suggests that (MRP6) ABCC6 has afunction-related to cellular detoxification which may affect thecalcification of elastic fibers in skin, arteries and the retina.Alternatively, calcification of elastic fibers in skin, arteries, andthe retina may result from MRP6 functional deficiencies in thosetissues.

The predicted structure of the MRP6 protein is shown in FIG. 2.Transmembrane domains (unshaded rectangles), nucleotide-binding foldregions (NBF) and Walker motifs are indicated and were identified byamino acid sequence homology with similar transporters. Arrows indicatethe positions of several PXE associated mutations. The large shadedrectangle represents the cell membrane.

According to the invention, the transmembrane domains of the MRP6protein shown in FIG. 2 are hydrophobic stretches of amino acidsidentified via transmembrane domain predictions (SOSUI and DAStransmembrane prediction programs). Regions of MRP6/ABCC6 with a highdegree of conservation when compared with similar proteins (ABCtransporters) include the regions involved in the binding and hydrolysisof ATP also known as nucleotide binding folds (NBF). According to theinvention, the MRP6 protein has two nucleotide-binding fold regions(NBF1 and NBF2) as shown in FIG. 2. These regions correspond to thefollowing amino acid segments of the human MRP6 protein: NBF1 residues656-679, 747-768, and 775-784 of SEQ ID NO: 3; and NBF2 residues1292-1307, 1321-1327, and 1403-1433 of SEQ ID NO: 3.

According to one embodiment of the invention, conserved amino acids inthe MRP6 protein are amino acids identified by comparing 1.2 ABCtransporter proteins from Human, Rat, Mouse, C. elegans, Yeast (S.cerevisiae) and A. thaliana. Preferred conserved amino acids are shownin FIG. 3 (conserved amino acids are underlined). According to theinvention, conserved domains are concentrated in the C-terminal portionof the protein, where over 90% of the PXE causing mutations have beenidentified.

ii) Mutations in the (MRP6) ABCC6 Gene

According to one aspect of the invention, PXE is caused by a mutation atthe (MRP6) ABCC6 locus that results in reduced MRP6 protein function.PXE associated mutations include mutations that affect the level of MRP6protein expression in addition to mutations that alter the functionalproperties of an expressed MRP6 protein PXE associated mutations at the(MRP6) ABCC6 locus include chain-terminating mutations. Such mutationsare typically recessive and account for the autosomal recessive natureof the associated PXE phenotype. However, PXE associated mutationsidentified at the (MRP6) ABCC6 locus include chain terminating mutationsat different positions in the (MRP6) ABCC6 gene, and severalsubstitution, deletion and insertion mutations. According to theinvention, the C-terminal half of the MRP6 protein is functionallyimportant. Indeed, many of the PXE associated mutations were identifiedin exons 23-29. However, even a I to T substitution at position 1424(out of 1503 amino acid residues) results in a PXE associated phenotype.Accordingly, a chain terminating or frameshift mutation in any one ofexons 1-29, even up to position 1424 in exon 30, and maybe even beyondis expected to be associated with PXE. According to the invention, thePXE phenotype associated with different mutations in the (MRP6) ABCC6gene varies in relation to the functional properties of the mutant(MRP6) ABCC6 protein product. Therefore, individuals with different PXEassociated mutations can have PXE symptoms of differing severity. Inaddition, different individuals having the same PXE mutations, but indifferent genetic backgrounds, can also develop PXE symptoms ofdiffering severity. Accordingly, different mutations at the PXE locusare expected to result in PXE phenotypes of differing severity. Forexample, in one embodiment of the invention, a mutation that results inthe absence of MRP6 protein expression (for example a deletion of partor all of the gene, a chain terminating mutation, a mutation thatprevents mRNA production, or a mutation that prevents translation of themRNA) is expected to have a more severe PXE phenotype than a mutationthat interferes with normal MRP6 protein function without destroying thefunction (for example an amino acid substitution that alters thestructure and function of the protein without inactivating it. Inparticular, an individual that is a homozygote for a mutation thatprevents MRP6 protein expression, or that is a compound heterozygotewith two different mutations each of which prevents MRP6 proteinexpression, is expected to have a more severe phenotype than anindividual that has a mutation with less severe effects on MRP6 proteinfunction at one or both alleles of the MRP6 locus.

In a further embodiment of the invention, a heterozygote carrier of aPXE mutation can exhibit characteristic manifestations of PXE. Inparticular, a carrier of a recessive mutation can show partial skin, eyeor cardiovascular symptoms. According to the invention, heterozygotecarriers of different (MRP6) ABCC6 mutations can develop differentsubsets of PXE related symptoms and can have symptoms of varyingseverity. Indeed, there are numerous examples of dermal “elastic fiberschanges” or cardiovascular abnormalities ranging from hypertension tomyocardial infarction, in family members of severely affectedindividuals. According to the invention, cases of partial expression ofPXE symptoms in heterozygote carriers are cases that had been assumed tobe examples of dominant inheritance with for example 10 to 20%penetrance.

The various subtypes of a disorder or a dual mode of inheritance of adisease are frequently due either to mutations in different genes ordifferent mutations in the same gene. Epidermolysis buflosa (EB) is anexcellent example of a disorder characterized by several clinical typescaused by distinct mutations in the same gene or mutations in differentgenes. EB is viewed as a group of heritable mechano-bullous skindiseases classified into three major categories of simplex, junctionaland dystrophic forms. EB simplex is due to mutations in the genesencoding keratins 5 and 14, the junctional form is associated withmutations in the kalininl7aminin 5 genes; and the dystrophic disorderresult from mutations in the type VII collagen gene (COL7Al). Thedystrophic EB presents clinical sub-types: the Hallopeau-Siemens type isautosomal recessive and caused by nonsense mutations and glycinesubstitutions result in the autosomal dominant form.

In contrast to EB, no locus heterogeneity has been shown for PXE.According to the invention, most cases of PXE, if not all, are due to(MRP6) ABCC6 mutations. While the clinical heterogeneity in PXE patientsmay be caused by different types of (MRP6) ABCC6 mutations, thedifferent PXE lesions (vascular, ocular, and dermal) observed fordifferent autosomal recessive and seemingly dominant PXE mutations areclinically indistinguishable. Furthermore, identical PXE mutations canbe either recessive or apparently dominant in unrelated pedigrees.Accordingly, different PXE mutations in different genetic backgroundsare associated with different severities of PXE symptoms. Furthermore, aPXE mutation can result in a partial PXE phenotype in a carrierindividual (thereby accounting for observations of apparent dominantforms of PXE).

iii) Population Distributions of (MRP6) ABCC6 Mutations

According to the invention, different PXE associated (MRP6) ABCC6mutations exist in the population, and new (MRP6) ABCC6 mutations arisesporadically. Based on current estimations of the prevalence of PXE inthe United States (between 1:100,000 and 1:25,000), the frequency ofappearance of heterozygote individuals with PXE mutations should bebetween 0.6 and 2.5 percent of the general population (1.5 to 6.0million individuals). Given the risk of heterozygote individuals havingchildren with PXE, an important aspect of the invention is to provide agenetic screen to identify heterozygote carriers of PXE mutations.According to the invention, a PXE carrier is an individual with onemutant allele of the (MRP6) ABCC6 gene, wherein the mutant allele is anallele that results in a PXE phenotype in an individual that ishomozygous for that allele (or in an individual that is heterozygouswith two different (MRP6) ABCC6 mutant alleles, each of which isassociated with PXE).

According to a further embodiment of the invention, a significant factorin the complex phenotype of the PXE multi-organ disorder is partialexpression of the full range of the PXE symptoms in heterozygotecarriers in recessive pedigrees. For example, a single mutant-ABCC6allele, for example R1141X, within heterozygote carriers can manifest apartial, mostly vascular-related phenotype. Indeed, cardiovascularabnormalities are frequently seen in obligate carriers but ocular anddermal lesions have also been diagnosed. The PXE phenotype, as observedin several heterozygous carriers, range from sub-clinical manifestationsto visible lesions. The spectrum of these partial phenotypes overlapswith that of the less severely affected PXE patients. There is,therefore, a continuum in the PXE phenotype between heterozygouscarriers and PXE patients, which make the clinical diagnosis of the lesssevere forms of PXE equivocal. According to the invention,cardiovascular symptoms associated with PXE mutations at the MRP6 geneinclude atherosclerosis, hypertension, stroke, gastrointestinalbleeding, intermittent claudication. Ocular symptoms include macular orretinal degeneration and skin related symptoms include premature agingand solar elastosis.

According to the invention, the identification of the PXE gene providesmethods for an unambiguous molecular diagnosis of patients and theidentification of heterozygous carriers in families with autosomalrecessive PXE or apparent autosomal dominant PXE, and the identificationof homozygous PXE individuals or PXE carriers in the general population.

According to the invention, different populations can contain differentcharacteristic PXE associated MRP6 mutations or different frequencies ofPXE associated MRP6 mutations due to factors such as founder effects.For example, a founder effect in the South African Afrikaner populationis thought to have caused the observed higher frequency of PXE inAfrikaners. According to the invention, a higher frequency of PXE in apopulation correlates with a higher frequency of PXE associated MRP6mutations.

Intra-familial variation of the phenotype is a well known characteristicof PXE. These variations may be due to genetic and/or environmentalcauses. A few environmental factors are thought to influence the PXEphenotype. Among these, calcium and Vitamin D have been reported tocontribute to the severity of the phenotype in some cases. Life style,smoking, diet, sun-exposure and obesity are also likely to modulate thepenetrance of the phenotype. Indeed, remarkably dissimilar PXEphenotypes have been observed recently in identical twins. According tothe invention, non-genetic factors contributing to the development ofPXE symptoms in heterozygote carriers can be identified. Studiesinvolving large cohorts of twins for example, such as those used by theQueensland Institute of Medical Research of Australia are also useful toidentify both genetic and environmental factors related to thedevelopment of the PXE phenotype.

II. Diagnostic Applications

(MRP6) ABCC6 genes and gene products, including mutant genes and geneproducts, as well as probes, primers, and antibodies, are useful foridentifying carriers of PXE associated mutations. According to theinvention, PXE associated mutations can be identified in families with aPXE pedigree or in individuals not previously known to be at risk ofcarrying a PXE related mutation. PXE associated mutations can beroutinely screened using probes to detect the presence of a mutant(MRP6) ABCC6 gene or protein by a variety of methods. In preferredembodiments of the invention, individuals are screened for the presenceof a recurrent mutation that is known to be present at a relatively highfrequency in the population. For example, a preferred method of theinvention screens an individual from a population for the presence of anMRP6 mutation that accounts for about 30%, and preferably 50%, and morepreferably over 50%, of known incidences of PXE in the population. Analternative method of the invention screens an individual for thepresence of two or more, preferably about five, more preferably aboutten, and even more preferably over ten PXE associated MRP6 mutations. Inmethods that include assays for a plurality of PXE associated MRP6mutations, the plurality of mutations preferably account for about 30%,and more preferably 50%, and even more preferably over 50%, of knownincidences of PXE in the population.

In one aspect of the invention, the identification of a specificmutation is not necessary. A diagnostic assay may be based on thedetection of an MRP6 protein expression defect resulting from, forexample, reduced levels of mRNA expression. Indeed, the analysis ofsteady state levels of (MRP6) ABCC6 mRNA in skin fibroblasts from a PXEpatient carrying a homozygous R1141X mutation showed that MRP6 mRNAlevels were lower than in skin fibroblasts from a normal individual.Accordingly, low levels MRP6 mNRA can result from a mutation within thecoding sequence, such as a nonsense mutation that results in nonsensemediated decay. In addition, low mRNA levels can be caused by mutationseither an intron or an exon that destabilizes the RNA, or by a mutationin a regulatory region (including a promoter region) that reducestranscription of the MRP6 gene. Furthermore, the presence of a truncatedMRP6 mRNA can be used as a diagnostic indicator for the presence of aPXE associated mutation.

Alternatively, the presence of a mutation that affects the amount, size,or other physical properties of the MRP6 protein can be detected withoutknowing the identity of the mutation. For example a decreased level ofMRP6 protein or a the presence of a truncation in the MRP6 protein canbe used as a diagnostic indicator for the presence of a PXE associatedmutation. In addition, the presence of a larger than expected MRP6protein (that may result for example, from a gene fusion or from one ormore frameshift mutations that produce a larger and possiblynon-functional protein) can be used as a diagnostic indicator for thepresence of a PXE associated mutation.

Accordingly the invention provides a method for screening for thepresence of a PXE associated mutation at the MRP6 locus withoutspecifically identifying the mutation. Such methods are useful toidentify homozygotes, compound heterozygotes, or carriers.

According to the invention, the identification of the presence of anyPXE associated mutation at the MRP6 locus can be used as a positivediagnosis of PXE in an individual with PXE symptoms or to diagnose a PXEpatient who has not yet developed PXE symptoms but who is identified asa homozygote or a compound heterozygote for PXE associated MRP6mutations. Alternatively, the detection of the presence of a PXEassociated MRP6 mutation according to the invention provides a methodfor screening a population to identify individuals who are carriers of aPXE associated mutation.

In general, a PXE carrier is distinguished from a PXE homozygote by thepresence of both a normal allele and a PXE mutant allele in the carrierand the presence of two PXE mutant alleles in the homozygote. Accordingto the invention, a normal allele can contain a neutral polymorphism asdisclosed herein.

a) Nucleic Acid Based Diagnostics

When a diagnostic assay is to be based upon nucleic acids from a sample,the assay may be based upon mRNA, cDNA or genomic DNA. If mRNA is usedfrom a sample, there may be little or no expression of transcriptsunless appropriate tissue sources are chosen or available. Preferredtissue sources are biopsies of full thickness skin or skin fibroblastscultured from dermal biopsies. Whether mRNA, cDNA or genomic DNA isassayed, standard methods well known in the art may-be used to detectthe presence of a particular sequence either in situ or in vitro (see,e.g., Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). As ageneral matter, however, any tissue with nucleated cells may beexamined.

Genomic DNA used for the diagnosis may be obtained from body cells, suchas those present in the blood, tissue biopsy, surgical specimen, orautopsy material. The DNA may be isolated and used directly fordetection of a specific sequence or may be amplified by the polymerasechain reaction (PCR) prior to analysis. Similarly, RNA or cDNA may alsobe used, with or without PCR amplification. To detect a specific nucleicacid sequence, direct nucleotide sequencing hybridization using specificoligonucleotides, restriction enzyme digest and mapping, PCR mapping,RNase protection, chemical mismatch cleavage, ligase-mediated detection,and various other methods may be employed. Oligonucleotides specific toparticular sequences can be chemically synthesized and labeledradioactively or non-radioactively (e.g., biotin tags, ethidiumbromide), and hybridized to individual samples immobilized on membranesor other solid-supports (e.g., by dot-blot or transfer from gels afterelectrophoresis), or in solution. The presence or absence of the targetsequences may then be visualized using methods such as autoradiography,fluorometry, or colorimetry. These procedures can be automated usingredundant, short oligonucleotides of known sequence fixed in highdensity to silicon chips, or in other oligonucleotide array formats.

Whether for hybridization, RNase protection, ligase-mediated detection,PCR amplification or any other standards methods described herein andwell known in the art, a variety of subsequences of the MRP6 sequencesdisclosed or otherwise enabled herein will be useful as probes and/orprimers. These sequences or subsequences will include both normal MRP6sequences and PXE associated MRP6 mutant sequences. In general, usefuloligonucleotide probes or primer sequences will include at least 8-9,more preferably 10-50, and most preferably 18-24 consecutive nucleotidesfrom the MRP6 introns, exons or intron/exon boundaries. Depending uponthe target sequence, the specificity required, and future technologicaldevelopments, shorter sequences may also have utility. Therefore, anyMRP6 derived sequence which is employed in a diagnostic assay may beregarded as an appropriate probe or primer. Particularly usefulsequences include nucleotide positions from the MRP6 gene for which PXE,associated mutations are known, or sequences which flank thesepositions.

As discussed above, a variety of PXE causing mutations have now beenidentified at the human MRP6 gene locus. Detection of these and otherPXE associated mutations is now enabled using isolated nucleic acidprobes or primers derived from normal or mutant MRP6 genes. According tothe invention, useful oligonucleotide probes or primers are derived fromsequences encoding the C-terminal half of the MRP6 protein, theconserved NBF sequences, and conserved amino acid sequence shown in FIG.3. Particularly useful oligonucleotides are derived from sequences knownto have PXE associated mutations, such as the sequences including themutations shown in Table 1. As disclosed above, a number of PXEassociated MRP6 mutations have already identified, and it is expectedthat more will be identified according to the compositions and methodsdisclosed herein. Therefore, the present invention provides isolatednucleic acid probes and primers corresponding to normal and mutantsequences from any portion of the MRP6 gene, including exons, introns,and 5′ and 3′ UTRs, which may be shown to be associated with thedevelopment of PXE.

Merely as an example, and without limiting the invention, usefuldiagnostic probes and primers derived from the MRP6 DNA are disclosed inExample 5.

For in situ hybridization-based detection of a normal or mutant MRP6, asample of tissue may be prepared by standard techniques and thencontacted with one or more of the nucleic acids described herein,preferably one which is labeled to facilitate detection, and an assayfor nucleic acid hybridization is conducted under stringent conditionswhich permit hybridization only between the probe and highly orperfectly complementary sequences. For the single nucleotidesubstitutions associated with PXE, high stringency hybridizationconditions will be required to distinguish most mutant sequences fromnormal sequences. When the MRP6 genotypes of an individual's parents areknown, probes may be chosen accordingly. Alternatively, probes to avariety of mutants may be employed sequentially or in combination.Because PXE carriers will be heterozygous, probes to normal sequencesalso may be employed and homozygous normal individuals may bedistinguished from mutant heterozygotes by the amount of binding (e.g.,by intensity of radioactive signal). In another variation, competitivebinding assays may be employed in which both normal and mutant probesare used but only one is labeled.

In addition to oligonucleotide-based hybridization assays, methods ofthe invention include direct sequencing, loss of heterozygosity, SSCP,HA, and Conformation-Sensitive Gel Electrophoresis (CSGE) to detect aPXE associated MRP6 mutation. As discussed above, preferred mutations tobe screened for are those shown in Table 1. However, additionalmutations identified according to the invention are also useful asmarkers of PXE, including deletions in the (MRP6) ABCC6 locus.

According to one embodiment of the invention, a diagnostic test can be anucleic scanning test where the assay detects the presence of a mutationin the nucleic acid being interrogated. In an alternative embodiment, adiagnostic test can interrogate a nucleic acid for the presence of aspecific mutation.

According to this invention, base pair deletions or alterations leadingto the omission of amino acid residues in the gene product aredetermined. Nucleic acid primers and probes are used in a variety ofPCR-based amplification and hybridization assays to screen for anddetect the presence of defective ABCC6 gene or mRNA in a patient. Thegenetic information derived from the intron/exon boundaries is also veryuseful in various screening and diagnosis procedures.

Various nucleic acid scanning methods are used for scanning the MRP6genomic, mRNA or cDNA sequence obtained from a patient for detecting,for example, large deletions and substitutions in the sequence thatwould be indicative of the disease. These nucleic acid scanningtechniques include PCR-based techniques and using oligonucleotide probesthat hybridize to specific regions of the gene.

In one embodiment of the invention, preferred mutations for nucleic acidscanning techniques include large deletions in the genomic sequence forthe ABCC6 gene for example a 16.5 kb deletion spanning from exon 22 toexon 29. Primers are designed to various regions of the ABCC6 gene whichare used for PCR-based detection of large deletions in the gene.

In another embodiment of the invention, primers are designed to theABCC6 gene that are able to differentiate between the size of theamplified wild-type sequence and sequence containing a specificmutation, for example a deletion.

In a preferred embodiment of the invention, nucleic acid probes areprovided which comprise either ribonucleic or deoxyribonucleic acids.Typically, the size of the probes varies from approximately 18 to 22nucleotides. Functionally, the probe is long enough to bind specificallyto the homologous region of the ABCC6 gene, but short enough such that adifference of one nucleotide between the probe and the DNA being testeddisrupts hybridization. Thus the nucleic acid probes of the presentinvention are capable of detecting single nucleotide changes in theABCC6 gene.

In a preferred embodiment of the invention, nucleic acid probes are 100%homologous to a mutant allele of the ABCC6 gene, but not to thewild-type gene.

In another embodiment of the invention, the nucleic acid probes are 100%homologous to the wild-type allele. Accordingly, the invention providesmethods for determining whether an individual is homozygous orheterozygous for a particular allele using both a wild-type and anallele-specific probe.

According to one method of the invention, mutations are detected bysequencing lo specific regions of the ABCC6 gene. In a preferredembodiment, the specific regions encompass one or more mutationspresented in Table 1. In an alternative embodiment, a specific regionbeing interrogated includes one of exons 1-31. Preferred exons includeexons 25-29, and more preferably exon 28 in which many PXE associatedmutations have been identified.

According to still other methods of the present invention rapidscreening techniques are used to determine whether exons of the ABCC6gene carry any mutations. Such techniques can be followed by one of thetechniques already described above which are specific for a particularallele or mutation. One such rapid screening technique involves thedetermination of the conformation of single strands of DNA which havebeen amplified from exon sequences that are known to carry mutations,including the mutations presented in Table 1. The single strands are runin non-denaturing electrophoretic gels, such as are typically used forsequencing DNA. The mobility of single stranded DNA on such gels issensitive to the conformation of the DNA fragments. The conformation ofthe single stranded DNA is dependent on its base sequence, alterationsin even one base affecting the conformation. Thus the presence of awild-type or mutant allele described herein can be detected byamplifying an exon sequence, denaturing the duplex molecules, andseparating them on the basis of their conformation on non-denaturingpolyacrylamide gels. If mutant alleles are present, they will have adifferent mobility than wild-type sequences amplified with the sameprimers. Most conveniently, the amplified sequences will be radiolabeledto facilitate visualization on gels. This can be readily accomplishedusing labeled primers or a labeled nucleotide. For a general referenceon this technique see Orira, et al., Genomics vol. 5, pp. 874-879(1989). A preferred nucleic acid amplification product for SSCP analysisis between about 100 and 500 bp, and more preferably between about 140and 300 bp.

According to another rapid screening technique of the present invention,an amplified fragment containing a mutation is detected using denaturinggradient gel electrophoresis (DGGE). For a general reference on thistechnique see Sheffield, et al., Proc. Natl. Acad. Sci. vol. 86, pp.232-236 (1989). Briefly, double stranded fragments which are generatedby amplification (PCR) can be subjected to DGGE. “DGGE is a gel systemthat separates DNA fragments according to their melting properties. Whena DNA fragment is electrophoresed through a linearly increasing gradientof denaturants, the fragment remains double stranded until it reachesthe concentration of denaturants equivalent to a melting temperature(Tm) that causes the lower-temperature melting domains of the fragmentto melt. At this point, the branching of the molecule caused by partialmelting sharply decreases the mobility of the fragment in the gel. Thelower-temperature melting domains of DNA fragments differing by aslittle as a single-base substitution will melt at slightly differentdenaturant concentrations because of differences in stackinginteractions between adjacent bases in each DNA strand. Thesedifferences in melting cause two DNA fragments to begin slowing down atdifferent levels in the gel, resulting in their separation from eachother.” Sheffield, et al., ibid. Use of a GC clamp as taught in Myers etal., Nucleic Acids Res. vol. 13, pp. 3111-3146 (1985) increases thesensitivity of detection of this method from about 40% to about 100%. Ifmismatches are present, which would be the case if the DNA sampleamplified was heterozygous for an ABCC6 allele, they will be visible onthese DGGE gels. Double stranded fragments containing one wild-typestrand and one mutant strand will have a different mobility on thesegels than will double stranded fragments which contain two wild-type ortwo mutant strands, due to the different melting temperatures of thesespecies. Thus, the melting temperature of fragments amplified fromdifferent regions of the ABCC6 gene can be determined by DGGE and can beused to indicate whether a mutant allele is present.

In one embodiment, a region of the (MRP6) ABCC6 gene that encodes animportant functional domain of the (MRP6) ABCC6 protein is screened forthe presence of any mutation. For example, a preferred diagnostic assayinterrogates the region of the (MRP6) ABCC6 gene that encodes an ATPbinding site of the (MRP6) ABCC6 protein, a region that encodes ahydrophobic transmembrane domain, or a region that encodes a conservedamino acid, preferably in the C-terminal half of the MRP6 protein.

One major application of the nucleic acid based diagnostics is in thearea of genetic testing, carrier detection and prenatal diagnosis.Individuals carrying mutations in the ABCC6 gene (disease carrier orpatients) may be detected at the DNA level with the use of a variety oftechniques. The genomic DNA used for the diagnosis may be used directlyfor detecting specific sequences or may be amplified enzymatically invitro, for example by PCR. The detection of specific DNA sequence may beachieved by methods such as hybridization using specificoligonucleotides (Wallace et al. Cold Spring Harbour Symp. Quant. Biol.51: 257-261 (1986)), direct DNA sequencing (Church and Gilbert, Proc.Nat. Acad. Sci. U.S.A. 81: 1991-1995 (1988)), the use of restrictionenzymes (Flavell et al. Cell 15: 25 (1978), Geever et al Proc. Nat.Acad. Sci. U.S.A. 78: 5081 (1981)), discrimination on the basis ofelectrophoretic mobility in gels with denaturing reagent (Myers andManiatis, Cold Spring Harbour Sym. Quant. Biol. 51: 275-284 (1986)),RNase protection (Myers, R. M., Larin, J., and T. Maniatis Science 230:1242 (1985)), chemical cleavage (Cotton et al Proc. Nat. Acad. Sci.U.S.A. 85: 4397-4401, (1985)) and the ligase-mediated detectionprocedure (Landegren et al Science 241:1077 (1988)).

Oligonucleotides specific to normal or mutant sequences are chemicallysynthesized using commercially available machines, labelledradioactively with isotopes or non-radioactively (with tags such asbiotin (Ward and Langer et al. Proc. Nat. Acad. Sci. U.S.A. 78:6633-6657 (1981)), and hybridized to individual DNA samples immobilizedon membranes or other solid supports by dot-blot or transfer from gelsafter electrophoresis. The presence or absence of these specificsequences are visualized by methods such as autoradiography orfluorometric (Landegren et al, 1989) or colorimetric reactions (Gebeyshuet al. Nucleic Acids Research 15:.4513-4534 (1987)).

Sequence differences between normal and mutants may be revealed by thedirect DNA sequencing method of Church and Gilbert. Cloned DNA segmentsmay be used as probes to detect specific DNA segments. The sensitivityof this method is greatly enhanced when combined with PCR (Wrichnik etal, Nucleic Acids Res. 15:529-542 (1987); Wong et al, Nature 330:384-386(1987); Stoflet et al, Science 239:491-494 (1988)). In the latterprocedure, a sequencing primer which lies within the amplified sequenceis used with double-stranded PCR product or single-stranded templategenerated by a modified PCR. The sequence determination is performed byconventional procedures with radiolabeled nucleotides or by automaticsequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing reagent. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. Forexample, a PCR product with a small deletion is clearly distinguishablefrom the normal sequence on an 8% non-denaturing polyacrylamide gel. DNAfragments of different sequence compositions may be distinguished ondenaturing formamide gradient gel in which the mobilities of differentDNA fragments are retarded in the gel at different positions accordingto their specific “partial-melting” temperature (Myers, supra). Inaddition, sequence alterations, in particular small deletions, may bedetected as changes in the migration pattern of DNA heteroduplexes innon-denaturing gel electrophoresis, as have been detected for the 3 dp(I507) mutation and in other experimental systems (Nagamine et al, Am.J. Hum. Genet, 45:337-339 (1989)). Alternatively, a method of detectinga mutation comprising a single base substitution or other small changecould be based on differential primer length in a PCR. For example, oneinvariant primer could be used in addition to a primer specific for amutation. The PCR products of the normal and mutant genes can then bedifferentially detected in acrylamide gels.

Sequence alterations may occasionally generate fortuitous restrictionenzyme recognition sites which are revealed-by the use of appropriateenzyme digestion followed by conventional gel-blot hybridization(Southern, J. Mol. Biol 98: 503 (1975)). DNA fragments carrying the site(either normal or mutant) are detected by their reduction in size orincrease of corresponding restriction fragment numbers. Genomic DNAsamples may also be amplified by PCR prior to treatment with theappropriate restriction enzyme; fragments of different sizes are thenvisualized under UV light in the presence of ethidium bromide after gelelectrophoresis.

In another embodiment of the invention, sequence changes at specificlocations may also be revealed by nuclease protection assays, such asRNase (Myers, supra) and S1 protection (Berk, A. J., and P. A. SharpeProc. Nat. Acad. Sci. U.S.A. 75: 1274 (1978)), the chemical cleavagemethod (Cotton, supra) or the ligase-mediated detection procedure(Landegren supra).

In addition to conventional gel-electrophoresis and blot-hybridizationmethods, DNA fragments may also be visualized by methods where theindividual DNA samples are not immobilized on membranes. The probe andtarget sequences may be both in solution or the probe sequence may beimmobilized (Saiki et al, Proc. Natl. Acad. Sci USA, 86:6230-6234(1989)). A variety of detection methods, such as autoradiographyinvolving radioisotopes, direct detection of radioactive decay (in thepresence or absence of scintillant), spectrophotometry involvingcolorigenic reactions and fluorometry involving fluorogenic reactions,may be used to identify specific individual genotypes.

In a preferred embodiment of the invention, for example, a PCR withmultiple, specific oligonucleotide primers and hybridization probes, maybe used to identify a plurality of possible mutations at the same time(Chamberlain et al. Nucleic Acids Research 16: 1141-1155 (1988)). Theprocedure may involve immobilized sequence-specific oligonucleotidesprobes (Saiki et al, supra).

According to the invention, assays are performed to detect a deletionwithin or including the MRP6 gene using Southern hybridization, FISHanalysis, or diagnostic PCR. It is expected that most deletions willoccur between repetitive Alu sequences that are common within theintrons of the MRP6 gene. Preferred PCR primers for detecting thesedeletions are primers that flank intron Alu sequences.

According to one aspect of the invention, many of the PXE associatedMRP6 mutations are found in exons 22-30. Accordingly, preferred assaysof the invention interrogate any one of exons 22-30, taken alone or incombination, for the presence of a PXE associated MRP6 mutation.

In a preferred embodiment of the invention, a diagnostic assayinterrogates the entire (MRP6) ABCC6 locus for the presence of amutation, using for example SSCP, HA, or CSGE, and direct sequencing. Ina more preferred embodiment, an assay interrogates a portion of theABCC6 locus for the presence of a mutation. If a mutation is detected,it is first compared to known mutations associated with PXE (Table 1)and known neutral polymorphisms (Table 2) that are not associated withPXE. If the mutation has not yet been observed as either a PXEassociated mutation or as a neutral polymorphism, the nature of themutation is considered. If the mutation is a deletion, nonsense,frameshift or other mutation that affects expression of a normal MRP6protein, the mutation is considered to be a PXE mutation. Similarly, ifthe mutation results in a nonconservative amino acid change or an aminoacid change in a conserved sequence such as an NBF, a transmembranesequence, or a change in a conserved amino acid shown in FIG. 3, themutation is considered to be a PXE mutation. In addition, if themutation results in low levels of MRP6 expression, the mutation isconsidered to be a PXE mutation. However, if the mutation results in aconservative amino acid change in a non-conserved part of the MRP6protein the mutation is considered to be a neutral polymorphism.Nonetheless, a patient identified with a previously unknown neutralpolymorphism according to this analysis should be subjected toadditional diagnostic tests to look for known PXE associated symptoms orsubclinical symptoms.

According to one aspect of the invention, the detection of PXE carriersand PXE patients is determined through the identification of mutant MRP6alleles in DNA from patients, family members and apparently unrelatedand normal individuals. A single allele, with no evidence of a secondmutant allele and the presence of a normal allele will be considered acarrier. Patients may be identified as either compound heterozygotes(having two different mutant alleles) or homozygotes (two identicalmutant alleles).

b) Protein Based Diagnostics

Different approaches to a MRP6 protein-based diagnostic assay can beused to detect the presence of a PXE related mutation in a patient.Preferred assays include detecting a mutant electrophoretic mobility,the presence of a mutant epitope, the absence of a normal epitope, or byidentifying altered biological activity, for example altered ATP bindingor altered transport of a synthetic, preferably radiolabeled molecule.

In one embodiment, diagnosis can be achieved by monitoring differencesin the electrophoretic mobility of normal and mutant proteins. Such anapproach will be particularly useful in identifying mutants in whichcharge substitutions are present, or in which insertions, deletions orsubstitutions have resulted in a significant change in theelectrophoretic migration of the resultant protein. Alternatively,diagnosis may be based upon differences in the proteolytic cleavagepatterns of normal and mutant proteins, differences in molar ratios ofthe various amino acid residues, or by functional assays demonstratingaltered function of the gene products.

In preferred embodiments, protein-based diagnostics will employdifferences in the ability of antibodies to bind to normal and mutantMRP6 proteins. Such diagnostic tests may employ antibodies which bind tothe normal proteins but not to mutant proteins, or vice versa. Inparticular, an assay in which a plurality of monoclonal antibodies, eachcapable of binding to a mutant epitope, may be employed. The levels ofanti-mutant antibody binding in a sample obtained from a test subject(visualized by, for example, radiolabelling, ELISA or chemiluminescence)may be compared to the levels of binding to a control sample.Alternatively, antibodies which bind to normal but not to mutant MRP6protein may be employed, and decreases in the level of antibody bindingmay be used to distinguish homozygous normal individuals from mutantheterozygotes or homozygotes. Such antibody diagnostics may be used forin situ immunohistochemistry using biopsy samples of tissues obtainedfrom patients.

c) Genetic Counseling

According to one embodiment of the invention, genetic counseling isprovided to an individual identified as a PXE carrier, a PXE homozygote,or a PXE compound heterozygote (an individual with two different PXEmutant alleles). According to the invention, individuals carrying twoPXE mutant alleles are provided information about amelioratingtreatments for some of the symptoms of PXE. For example, a person whoinherits PXE recessively is cautioned with regard to diet and activity.A low fat, high fibre, heart healthy diet is critical for maintainingcardiovascular health. Regular exercise appears to alleviate some of thesymptoms of peripheral vascular disease. Medications to allow thepassage of blood through narrowed arteries may be recommended.Individuals exhibiting eye manifestations should not engage inactivities that put them at risk for injury to the eye that couldsubsequently lead to hemorrhage and vision loss. Smoking should beavoided at all costs since it appears to increase the rate and severityof eye disease. In one embodiment of the invention, a patient identifiedas being a carrier of a PXE associated mutation or as being a homozygoteor a compound heterozygote for PXE associated mutations should beadvised to reduce calcium intake or to use drugs that reduce calciumintake in order to reduce the severity of the phenotype.

III. Therapeutic Applications

The present invention provides a basis for therapeutic treatments of PXErelated symptoms caused by mutations at the (MRP6) ABCC6 locus.According to the invention, normal (MRP6) ABCC6 nucleic acid or proteinis provided to cells and/or a patient having a PXE associated mutationat the (MRP6) ABCC6 locus.

Preferred target tissues include the kidney and liver, but also othertissues where low levels of MRP6 expression have been observed, such assmooth muscle cells arid macrophages. Preferred target tissues alsoinclude tissues or cells that exhibit PXE related symptoms, such as ablood vessel, the gastrointestinal tract, occular tissue, the urinarytract, and skin.

a) Nucleic Acid-based Therapeutics

According to the invention, PXE or PXE associated symptoms can beprevented or treated by providing a normal PXE gene or cDNA to a patientthat is diagnosed as having on or more PXE associated mutations at the(MRP6) ABCC6 locus. The fact that PXE is a recessive disease makes itparticularly amenable to gene therapy, because it is expected that most,if not all, PXE associated MRP6 mutations reduce the amount offunctional MRP6 protein in a cell and can be compensated for byproviding normal MRP6 to the cell.

In one series of embodiments, normal copies of the MRP6 gene areintroduced into patients to code successfully for normal protein in oneor more different affected cell types. The gene must be delivered tothose cells in a form in which it can be taken up and code forsufficient protein to provide effective function. Thus, it is preferredthat the recombinant gene be operably joined to a strong promoter so asto provide a high level of expression which will compensate for theabsence of sufficient amounts of normal MRP6. As noted above, therecombinant construct may contain endogenous or exogenous regulatoryelements, inducible or repressible regulatory elements, ortissue-specific regulatory elements.

Preferred vectors for introducing an MRP6 gene to a cell or a patientinclude retroviral vectors, because of their high efficiency ofinfection and stable integration and expression. Other viral vectorswhich can be used include adeno-associated virus, vaccinia virus, bovinepapilloma virus, or a herpes virus such as Epstein-Barr virus.Alternative vectors include plasmids that are replicated in human cells.

In another series of embodiments, a mutant MRP6 gene may be replaced byhomologous recombination with a recombinant construct. The recombinantconstruct preferably contains a normal copy of the MRP6 gene.Alternatively, a regulatory region of a normal MRP6 gene in a PXEcarrier may be altered to increase expression of normal MRP6.

i) Wild Type Genes

In one series of embodiments, a normal human (MRP6) ABCC6 gene isintroduced to cells or a patient. A normal (MRP6) ABCC6 gene includes agene with one or more polymorphic variations that are not associatedwith PXE. In one embodiment, an MRP6 genomic sequence is used. In analternative embodiment an MRP6 cDNA sequence is used.

ii) Related Genes

In an alternative series of embodiments, an (MRP6) ABCC6 related gene isprovided to a cell or tissue having a PXE associated mutation. Accordingto the invention, an (MRP6) ABCC6 related gene encodes a protein thathas similar functional properties as a normal human MRP6 protein and cancompensate for the absence of sufficient amounts of normal human MRP6protein in a patient cell or tissue. Preferably, an (MRP6) ABCC6homologue from another mammalian species is used. For example the mouseor rat MRP6 genes or cDNAs could be used. In one embodiment of theinvention, a homologue from a non-mammalian species is used.Alternatively, a nucleic acid encoding a different ABC protein is used,for example an MRP1 encoding nucleic acid.

The present invention also provides for cells or cell lines, bothprokaryotic and eukaryotic, which have been transformed or transfectedwith the nucleic acids of the present invention so as to cause clonialpropagation of those nucleic acids and/or expression of the proteins orpeptides encoded thereby. Such cells or cell lines will have utilityboth in the propagation and production of the nucleic acids and proteinsof the present invention but also, as further described herein, as modelsystems for diagnostic and therapeutic assays. As used herein, the term“transformed cell” is intended to embrace any cell, or the descendant ofany cell, into which has been introduced any of the nucleic acids of theinvention, whether by transformation, transfection, infection, or othermeans. Methods of producing appropriate vectors, transforming cells withthose vectors, and identifying transformants are well known in the art.

b) Protein Based Therapeutics

Treatment of PXE symptoms may be performed by directly providing normalprotein to a patient cell or tissue. Sufficient amounts of substantiallypure MRP6 protein can be obtained from cultured cell systems whichexpress the protein. Delivery of the protein to the affected tissue canthen be accomplished using appropriate packaging or administratingsystems including, for example, liposome mediated protein delivery tothe target cells.

c) Drug Therapies

In one embodiment of the invention, a drug identified according tomethods of the invention is administered to a patient diagnosed with PXEor a PXE carrier with PXE related symptoms. Alternatively, a drug isadministered to prevent or minimize the development of PXE or PXEassociated symptoms in individuals identified as having one or more PXEmutations at the ABCC6 locus.

IV. Drug Discovery Applications

The present invention provides a basis for screening drug candidates toidentify useful therapeutic compositions to treat or alleviate thesymptoms of PXE. In a series of embodiments, the invention providesscreens based on MRP6 activity. As used with respect to this series ofembodiments, the term “activity” broadly includes gene and proteinexpression, protein post-translation processing, trafficking andlocalization, and any functional activity (e.g., enzymatic,receptor-effector, binding, channel), as well as downstream effects ofany of these. MRP6 appears to be an integral membrane protein and mayhave transport related functions, and it also has ATP binding cassettes.Accordingly, these functional properties can be used as a basis for ascreen to identify compounds that increase MRP6 function.

In one embodiment, a drug candidate is screened for its ability toincrease expression of the MRP6 gene. A preferred screen monitors thelevel of normal MRP6 mRNA in cells grown in culture in the presence andabsence of the candidate compound. Alternatively, normal MRP6 proteinlevels are monitored. Useful cells for these assays are preferablynormal cells or PXE carrier cells. However, a PXE cell can also be usedand the levels of mutant MRP6 expression can also be monitored. Acompound that increases the level of MRP6 expression is particularlyuseful to treat a PXE carrier in order to increase the level of MRP6expressed from the normal allele. However, a compound that increases thelevel of MRP6 expression can also be useful to treat a PXE homozygote orcompound heterozygote if the PXE associated MRP6 allele(s) encodes anMRP6 protein that retains some normal MRP6 function or if the allele isa mutation that reduces the level of normal MRP6 function.

Other assays are useful for screening candidate compounds to identify acompound that increases normal MRP6 function. In one embodiment, anassay screens a compound for the ability to restore normal phenotype todermal fibroblasts isolated from a PXE patient. Dermal fibroblastsisolated from patients with PXE exhibit abnormal phenotype when grown invitro (Quaglino et al., Biochimica et Biophysica Acta 1501 (2000)51-62). These phenotypes include an increased proliferation indexcompared to normal fibroblasts when grown in monolayer. PXE fibroblastsalso have lower adhesion properties to collagen type I and to plasmafibronectin when compared to normal fibroblasts. Accordingly, thesephenotypes provide a basis for an assay to identify a compound thatrestores normal MRP6 function to dermal fibroblasts isolated from apatient that was identified as having a PXE associated MRP6 mutation.

In another embodiment of the invention, an assay is used to screencandidate compounds for their ability to increase the ATPase activity ofan MRP6 proteins. In a preferred embodiment, the assay monitors theATPase activity of all MRP6 protein encoded by an MRP6 gene with a PXEassociated mutation in the presence and absence of the candidatecompound. ATPase activity of purified MRP6 can be assayed according tomethods known in the art (see, for example, Mao et al., Biochimica etBiophysica Acta 1461, 69-82 (1999). According to the invention, acompound that increases the ATPase activity of a PXE associated MRP6protein variant is useful to treat a patient that is heterozygous orhomozygous for the PXE allele that encodes the protein variant used inthe assay.

In a similar embodiment of the invention, an assay is used to screencandidate compounds for their ability to increase the transportactivities of an MRP6 protein, in particular a PXE associated MRP6protein variant. A useful transport assay is provided in Oude et al.,Biochim Biophys Acta, 1241(2), 215-68, 1995. A compound identifiedaccording to this screen is useful to treat PXE patients and PXEcarriers as described above.

V. Disease Models

The invention provides a basis for designing cellular and animal modelsof PXE. Such models are useful to study the development of the PXEdisease in PXE homozygotes and compound heterozygotes and to identifypotential PXE associated physiological dysfunctions in PXE carriers.Such models are also useful in screens to identify therapeutic compoundsto prevent or treat PXE symptoms.

a) Cellular Models

According to the invention, cellular models can be made by deleting oneor both MRP6 alleles, or by introducing one or more PXE associated MRP6alleles into a cell line grown in vitro, using methods known in the art.Preferred cell lines include renal and hepatic cell lines. Other usefulcell lines include those derived from skin (keratinocytes andfibroblasts) and ocular tissue (ganglioma cells).

b) Animal Models

The present invention also provides for the production of transgenicnon-human animal models for the study of PXE, for the screening ofcandidate pharmaceutical compounds, and for the evaluation of potentialtherapeutic interventions.

Animal species which suitable for use in the animal models of thepresent invention include, but are not limited to, rats, mice, hamsters,guinea pigs, rabbits, dogs, cats, goats, sheep, pigs, and non-humanprimates (e.g., Rhesus monkeys, chimpanzees). For initial studies,transgenic rodents (e.g., mice) are preferred due to their relative easeof maintenance and shorter life spans. Transgenic yeast or invertebrates(e.g., nematodes, insects) may be preferred for some studies becausethey will allow for even more rapid and inexpensive screening.Transgenic non-human primates, however, may be preferred for longer termstudies due to their greater similarity to humans.

Based on the identification of MRP6 as the gene associated with PXE,there are now several available approaches for the creation of atransgenic animal models for PXE, including animal models with one orboth MRP6 alleles deleted and animal models with one or two MRP6 alleleswith mutations similar to known PXE associated human MRP6 mutations.

To create an animal model (e.g., a transgenic mouse), a mutant MRP6 genecan be inserted into a germ line or stem cell using standard techniquesof oocyte microinjection, or transfection or microinjection intoembryonic stem cells. Animals produced by these or similar processes arereferred to as transgenic. If the mutation knocks out the MRP6 gene or aportion thereof, the animals are referred to as knockouts.

For oocyte injection, one or more copies of the recombinant DNAconstructs of the present invention may be inserted into the pronucleusof a just-fertilized oocyte. This oocyte is then reimplanted into apseudo-pregnant foster mother. The liveborn animals are screened forintegrants using analysis of DNA (e.g., from the tail veins of offspringmice) for the presence of the inserted recombinant transgene sequences.The transgene may be either a complete genomic sequence injected as aYAC, BAC, PAC or other chromosome DNA fragment, a cDNA with either thenatural promoter or a heterologous promoter, or a minigene containingall of the coding region and other elements found to be necessary foroptimum expression.

Retroviral infection of early embryos can also be done to insert therecombinant DNA constructs of the invention. In this method, thetransgene is inserted into a retroviral vector which is used to infectembryos (e.g., mouse or non-human primate embryos) directly during theearly stages of development to generate chimeras, some of which willlead to germline transmission.

Homologous recombination using stem cells allows for the screening ofgene transfer cells to identify the rare homologous recombinationevents. Once identified, these can be used to generate chimeras byinjection of blastocysts, and a proportion of the resulting animals willshow germline transmission from the recombinant line. In a preferredembodiment, inactivation of the MRP6 gene in mice may be accomplished bydesigning a DNA fragment which contains sequences from an MRP6 exonflanking a selectable marker. Homologous recombination leads to theinsertion of the marker sequences in the middle of an exon, causinginactivation of the MRP6 gene and/or deletion of internal sequences. DNAanalysis of individual clones can then be used to recognize thehomologous recombination events.

The techniques of generating transgenic animals, as well as thetechniques for homologous recombination or gene targeting, are nowwidely accepted and practiced. A laboratory manual on the manipulationof the mouse embryo, for example, is available detailing standardlaboratory techniques for the production of transgenic mice (Hogan etal., 1986). A large number vectors are available to accomplish this andappropriate sources of genomic DNA for mouse and other animal genomes tobe targeted are commercially available from companies such asGenomeSystems Inc. (St. Louis, Mo., USA). The typical feature of thesetargeting vector constructs is that 2 to 4 kb of genomic DNA is ligated5′ to a selectable marker (e.g., a bacterial neomycin resistance geneunder its own promoter element termed a “neomycin cassette”). A secondDNA fragment from the gene of interest is then ligated downstream of theneomycin cassette but upstream of a second selectable marker (e.g.,thymidine kinase). The DNA fragments are chosen such that mutantsequences can be introduced into the germ line of the targeted animal byhomologous replacement of the endogenous sequences by either one of thesequences included in the vector. Alternatively, the sequences can bechosen to cause deletion of sequences that would normally reside betweenthe left and right arms of the vector surrounding the neomycin cassette.The former is known as a knock-in, the latter is known as a knock-out.Example 5 describes a knockout of most of exons 28 and 29 in mouse MRP6.

VI. (MRP6) ABCC6 Interacting Molecules

According to the invention, molecules that interact with a normal MRP6gene product gene product provide candidates 1) for identifyingadditional types of mutations that result in a PXE phenotype and 2) foradditional levels of therapeutic intervention to overcome or minimizethe effect of a mutant PXE gene product. For example, the identificationof a protein that interacts with a normal MRP6 protein but not with aPXE mutant protein provides a potential target for therapeuticintervention if the function of the interacting protein can be modifiedto compensate for the absence of normal MRP6 protein.

According to the invention, (MRP6) ABCC6 interacting molecules can beidentified according to a number of biochemical and genetic methodsknown in the art, including affinity chromatography, mutationalanalysis, and yeast two hybrid analysis. As will be obvious to one ofordinary skill in the art, there are numerous other methods of screeningindividual proteins or other compounds, as well as large libraries ofproteins or other compounds (e.g., phage display libraries and cloningsystems from Stratagene, La Jolla, Calif.) to identify molecules whichbind to normal or mutant MRP6 proteins. All of these methods comprisethe step of mixing a normal or mutant MRP6 protein or protein fragmentwith test compounds, allowing for binding (if any), and assaying forbound complexes.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Materials and Methods

a) Sources of Patient Samples

PXE International, Inc.

To date, PXE International has assembled a database of over 2100 PXEpatients from 1400 families from 31 countries including North America,several European and South American countries and South Africa. Fromthis cohort of patients and family members, genomic DNA has beenprepared from whole blood samples obtained from over 1200 PXE patientsand family members.

Honolulu Heart Program

In the early 1950's, studies around the world were reporting geographicdifferences in coronary heart disease (CHD) mortality, pathology,prevalence, and incidence. Among these reports were those of significantdifferences in the CHD and cerebrovascular disease rates in Japan and inthe United States. The overall mortality for men in Japan and in theUnited States was similar, but the rates for CHD were strikinglydifferent. Reported CHD mortality among Japanese was approximatelytwenty percent of that among U.S. Caucasians. At about the same time,Japanese living in Hawaii and California were reported to have a loweroverall mortality than either U.S. Caucasians or Japanese living inJapan. The reasons for these differences were not apparent. However, itwas felt that the study of these populations might offer important cluesto the etiology of heart and vascular disease. The compared populations,living in Japan, Hawaii and California were of Japanese ancestry tolimit genetic variation between study groups. The Honolulu cohort of theNi-Hon-San (Nippon-Honolulu, San Francisco) study formed the basis forthe Honolulu Heart Program (HHP). That study has now been underway for35 years, providing extensive information about the role of lifestyle,diet, and other risk factors to development of chronic diseases of majorpublic health importance in 8,000 Japanese-American men.

Although several studies have suggested that PXE is more frequent infemales there is no evidence that the observed gender difference iscaused by genetic factors. Indeed, men would often report skin lesions,usually the first signs of PXE, later in life, than women. Therefore, astudy of an exclusively male cohort from the HHP should not compromisethe general applicability of the conclusions. PXE has also no particularpredilection for any ethnic or racial group. PXE has indeed largely beendescribed in Caucasians but also in African and Asian populations. PXEcases reported in Japan have shown no phenotypic or prevalencicdifferences when compared to those observed in Caucasian populations.

Unaffected Control Subjects

DNA has been prepared from 150 unrelated individuals with no evidence ofPXE. These samples have been aliquoted and are currently stored at −80°C. They are routinely used as control DNA samples and will be used toconfirm that any new and potential mutation detected in a PXE patient orrelative is indeed a mutation and not a neutral polymorphism. The donorsof these DNA samples were adults of either sex from various ethnicbackgrounds.

b) Mutation Detection Methods

Detection of Single Nucleotide Mutations

Single strand conformation polymorphism (SSCP) analysis is based on theobservation that single stranded DNA will adopt, in non-denaturingconditions, a secondary structure that is strictly sequence-dependant.Slight variations in sequence, such as a single nucleotide change canalter the conformation of a DNA fragment, which can be resolved on anon-denaturing polyacrylamide gel. Heteroduplex analysis (HA) is basedon the observation that heteroduplexes formed between two DNA strandswith one or more mismatches have electrophoretical mobility distinctlydifferent from homoduplexes. While both methods (SSCP and HA) can detectpoint mutations, some sequence variants are more readily detected by oneprocedure than the other. Accordingly, a preferred screening method,uses a combination of SSCP and modified HA called Conformation-SensitiveGel Electrophoresis or CSGE.

In a preferred assay, each characterized PCR primer pair isradioactively labeled sing T4 polynucleotide kinase and γ-[P³²]-ATP. ForSSCP analysis, radiolabeled PCR products are mixed with denaturingloading buffer and loaded onto a 0.5×MDE (MDE is a mutation detectionenhancement polyacrylamide-derived matrix provided by FMC products),0.6×TBE native polyacrylamide gel and electrophoresed overnight at 8watts in a sequencing gel apparatus. Separated, radiolabeled conformersare visualized by autoradiography. For CSGE, EDTA is added to theincubated PCR reaction mix to a final concentration of 1 mM and thereaction will be heat-denatured and incubated for 60 minutes at 68° C.to allow heteroduplex formation. Heteroduplex products are analyzed on a6% polyacrylamide gel (29:1 ratio of acrylamide/bisacrylamide), 10%(v/v) ethylene glycol and 15% (w/v) formamide in 0.5×TTE buffer (1×TTEis 89 mM Tris, 15 mM taurine, 0.5 mM EDTA, pH 9.0). A solution of 20%(v/v) ethylene glycol, 30% (w/v) formamide and 0.05% xylene cyanol andbromophenol blue is mixed equally with the samples. The gel is run at 35to 45 watts for 2 to 4 hours at room temperature. As for SSCP, CSGEconformers are revealed by autoradiography.

When an abnormal conformer or heteroduplex is detected, the segregationof the variant is analyzed for DNA samples from the entire family.Subsequently, the DNA sequence of the variant is determined by elutingnormal and altered DNA conformers directly from the electrophoresis gel.These PCR fragments are eluted in water, re-amplified and directly usedas a template for sequencing using an ABI 310 automated sequencer(Perking Elmer). A panel of 150 DNA samples of normal unrelatedindividuals is used to identify abnormal variants that are commonpolymorphisms in the ABCC6 locus.

Mutation Detection by Enzymatic Cleavage

Single nucleotide substitutions often modify the recognition site of arestriction enzyme. Polymorphisms and mutations can, therefore, bedetected in a rapid and convenient manner by the enzymatic cleavage of aPCR fragment containing the nucleotide change. This method is frequentlyemployed to verify the presence of previously characterized mutations orpolymorphisms in DNA samples for control, study or diagnostic purposes.It can also be used for screening a large number of samples. Out of theABCC6 mutations listed in Table 1, 10 mutations were identified with aunique restriction pattern. For example, three possible HhaI restrictionprofiles for one of these mutations. R1339C (4015C to T) can bevisualized by electrophoresis. According to the invention, singlenucleotide mutations in ABCC6 are detectable by enzymatic cleavage.Accordingly, this method is useful as an initial step to appropriatelycomplement the screening of large cohorts with more traditionalmutations detection techniques.

PCR Mapping

A single base substitution mutation may be detected based ondifferential PCR product length or production in PCR. Thus, primerswhich span mutant sites or which, preferably, have 3′ termini atmutation sites, may be employed to amplify a sample of genomic DNA, mRNAor cDNA from a subject. A mismatch at a mutational site may be expectedto alter the ability of the normal or mutant primers to promote thepolymerase reaction and, thereby, result in product profiles whichdiffer between normal subjects and heterozygous and/or homozygous MRP6mutants. The PCR products of the normal and mutant gene may bedifferentially separated and detected by standard techniques, such aspolyacrylamide or agarose gel electrophoresis and visualization withlabeled probes, ethidium bromide or the like. Because of possiblenon-specific priming or readthrough of mutation sites, as well as thefact that most carriers of mutant alleles will be heterozygous, thepower of this technique may be low.

Electrophoretic Mobility

Genetic testing based on DNA sequence differences also may be achievedby detection of alterations in electrophoretic mobility of DNA, mRNA orcDNA fragments in gels. Small sequence deletions and insertions, forexample, can be visualized by high resolution gel electrophoresis ofsingle or double stranded DNA, or as changes in the migration pattern ofDNA heteroduplexes in non-denaturing gel electrophoresis. MRP6 mutationsor polymorphisms may also be detected by methods which exploit mobilityshifts due to single-stranded conformational polymorphisms (SSCP)associated with mRNA or single-stranded DNA secondary structures.

Chemical Cleavage of Mismatches

Mutations in MRP6 may also be detected by employing the chemicalcleavage of mismatch (CCM) method. In this technique, probes (up to ˜1kb) may be mixed with a sample of genomic DNA, cDNA or mRNA obtainedfrom a subject. The sample and probes are mixed and subjected toconditions which allow for heteroduplex formation (if any). Preferably,both the probe and sample nucleic acids are double-stranded, or theprobe and sample may be PCR amplified together, to ensure creation ofall possible mismatch heteroduplexes. Mismatched T residues are reactiveto osmium tetroxide and mismatched C residues are reactive tohydroxylamine. Because each mismatched A will be accompanied by amismatched T, and each mismatched G will be accompanied by a mismatchedC, any nucleotide differences between the probe and sample (includingsmall insertions or deletions) will lead to the formation of at leastone reactive heteroduplex. After treatment with osmium tetroxide and/orhydroxylamine to modify any mismatch sites, the mixture is subjected tochemical cleavage at any modified mismatch sites by, for example,reaction with piperidine. The mixture may then be analyzed by standardtechniques such as gel electrophoresis to detect cleavage products whichwould indicate mismatches between the probe and sample.

Other Methods

Various other methods of detecting MRP6 mutations, based upon the MRP6sequences disclosed and otherwise enabled herein, will be apparent tothose of ordinary skill in the art. Any of these may be employed inaccordance with the present invention. These include, but are notlimited to, nuclease protection assays (S1 or ligase-mediated), ligatedPCR, denaturing gradient gel electrophoresis (DGGE), restrictionendonuclease fingerprinting combined with SSCP (REF-SSCP), and the like.

Methods for Analyzing MRP6 mRNA Levels:

The steady state levels of (MRP6) ABCC6 mRNA was analyzed in skinfibroblasts from a PXE patient carrying a homozygous R1141X mutation.Total skin fibroblast RNA from an unaffected control individual and aPXE patient was used to synthesize single stranded cDNA using oligo(dT).PCR primers derived from (MRP6) ABCC6 mRNA sequence were then used intwo consecutive rounds of 25 cycles of PCR. Poly(A)+ RNA from normalhuman kidney (obtained from Clontech) was used as a positive control fordetection of (MRP6) ABCC6 mRNA. MRP-1 mRNA was detected in the same cDNAsamples used for ABCC6 mRNA with 30 cycles of PCR. The 390 bp and 180 bpDNA fragments detected correspond to the expected size of (MRP6) ABCC6and MRP-1 mRNA domains encoded within exons 6-9 and 2-3 of the (MRP6)ABCC6 and MRP-1 genes respectively. No reverse transcriptase (No RT)controls were included to confirm that no PCR products were obtained inthe absence of cDNA synthesis.

Stringent Hybridization Conditions

High stringency conditions are at least equivalent to a temperature inthe range of about 40-70 degrees C., and between about 0.05 and 0.5 Msodium ion. High stringency hybridization conditions are well known inthe art and can be optimized for a specific oligonucleotide based on thelength and GC content of the oligonucleotide as described in, forexample, Sambrook et al., Molecular Cloning, A Laboratory Manual (ColdSpring Harbor, N.Y., 1982).

An oligonucleotide selected for hybridizing to the target nucleic acid,whether synthesized chemically or by recombinant DNA methodologies, isisolated and purified using standard techniques and then preferablylabeled (e.g., with ³⁵S or ³²P) using standard labeling protocols. Asample containing the target nucleic acid then is run on anelectrophoresis gel, the dispersed nucleic acids transferred to anitrocellulose filter and the labeled oligonucleotide exposed to thefilter under stringent hybridizing conditions, e.g. 50% formamide,5×SSPE, 2× Denhardt's solution, 0.1% SDS at 42° C., as described inSambrook et al. (1989) supra. The filter may then be washed using2×SSPE, 0.1% SDS at 68° C., and more preferably using 0.1×SSPE, 0.1% SDSat 68° C. Other useful procedures known in the art include solutionhybridization, and dot and slot RNA hybridization. Optionally, theamount of the target nucleic acid present in a sample is thenquantitated by measuring the radioactivity of hybridized fragments,using standard procedures known in the art.

Example 2 The Positional Cloning of the PXE Gene

Blood samples and skin biopsies from PXE patients and unaffectedrelatives in the United States ere collected by PXE International Inc.,by Dr. Ivonne Pasquali-Ronchetti in Italy, by Dr. F. Michael Pope in theUnited Kingdom and by Dr. Anne de Paepe in Belgium. Blood samples wereobtained from 100 Caucasian control individuals with no family historyof PXE. Genomic DNA was isolated from aliquots of blood. Low passage andconfluent skin fibroblasts were obtained from 3 mm full thickness skinbiopsies using known procedures.

To identify the gene that contains mutations responsible for PXE, thedisease locus was confined to a region of about 8 cM between markersD16S500 and- D16S3041. Recombination mapping reduced this large criticalregion to an 820 kb domain containing six candidate genes. These genesencode an isoform of Myosin Heavy Chain (MYH11), two MultidrugResistance-associated Proteins (MRP-1 and (MRP6) ABCC6), an unknownprotein called pM5 and two identical unknown proteins referred to asUNK. Using a polymorphic microsatellite repeat (GAAA₁₇) located at the5′ end of the MRP-1 gene (FIG. 1) an informative meiotic recombinationin one PXE patient was identified and this permitted the exclusion ofthe MYHH11 as a candidate gene and reduced the size of the PXE region to570 kb and 5 candidate genes. FIG. 1 shows the previously defined PXElocus covering 820 kb between markers D16S3060 and D16S79 at 16p13.1.The BAC contig that covers this region is shown along with the identityof the BACs. FIG. 1 b shows the gene content of the PXE locusrepresented from the telomere (left) to the centromere (right). Thetranscriptional orientation of the genes is indicated by arrows. A flagrepresents the position of a polymorphic marker (GAAA₁₇) used toidentify an additional meiotic recombination in one PXE patient thatexcluded the MYHII gene as the PXE gene.

The 109 exons within the five candidate genes were then screened formutations by Single-Strand Conformation Polymorphism (SSCP) andHeteroduplex Analysis (HA) using genomic DNA from a cohort of 20unrelated PXE patients.

Mutation detection, sequence analysis and RT-PCR, SSCP, and HeteroduplexAnalysis (HA) were carried out as previously described. Intron-derivedprimers for PCR amplification of exons present in the genes encodingMRP-1, (MRP6) ABCC6, pM5 and both UNK gene were synthesized using intronsequences available in the TIGR database. PCR products were typically150-350 bp in length and included complete intron/exon boundaries.Typical PCR reactions, were performed in the presence of.sup.32P-labelled primers in a 9700 thermocycler (Perkin Elmer).Radioactive PCR products were analyzed either by SSCP or HA using MDEpolyacrylamide gel (FMC) according to the manufacturer's instructions.DNA conformers were eluted in water from gel slices, re-amplified andsequenced utilizing the same primers used to generate these PCRproducts. DNA sequence analysis was performed using ABI BigDyeterminator cycle sequencing with an ABI310 automated DNA sequencer. Thesequence information generated by the sequencer was analyzed using theABI software. The Sequencher™ 3.1 program (Gene Codes Corporation, AnnArbor, Mich.) was used to identify variation between the sequence ofputative mutations and control sequences. RT-PCR was performed on totalRNA from cultured human skin fibroblasts and human kidney poly(A)+ RNA.The sequences of the PCR primers used are: (MRP6) ABCC6:5′-AGCCACGTTCTGGTGGGTTT-3′ (SEQ ID NO: 4); 5′-GGAGGCTTGGGATCACCAAT-3′(SEQ ID NO: 5); MRP-1: 5-CAACTGCATCGTTCTGTTTG-3′ (SEQ ID NO: 6); and5′-ATACTCCTTGAGCCTCTCCA-3′ (SEQ ID NO: 7). Following synthesis, PCRproducts were separated by electrophoresis through 1.2% agarose andvisualized by staining with ethidium bromide.

DNA sequence analysis of two conformers detected in PCR productscontaining exons 19 and 28 of the pM5 gene revealed two private singlenucleotide polymorphisms (SNPs) within the intronic sequence flankingthese exons (Table 2). These were the only sequence variants detected inthe 31 exons of the pM5 gene using a cohort of 20 PXE patients.Screening all eight exons of each of the two UNK genes revealed only oneSNP in the first exon of either one or both UNK genes in, 5 PXEpatients. This was a silent nucleotide change (C33T) within the 11^(th)codon (S11) of the open reading frame of either one or both unknowngenes and therefore unrelated to PXE. Screening all 31 exons of theMRP-1 gene in a panel of PXE patients identified several sequencevariants (Table 2) that are not functionally related to PXE as theyeither occurred in intronic sequences or did not encode changes in aminoacids. In addition, two missense variants (R633Q and G671V) were seen inexons-14 and 16 in two unrelated PXE patients but these substitutionswere unlikely to be responsible for PXE as they were also found in apanel of 200 alleles from unaffected, ethnically matched controlindividuals.

Finally, in screening the 31 exons of the (MRP6) ABCC6 gene, the firstmutations that are clearly responsible for PXE were identified. A C->Tsubstitution within exon 24 (C3421T) of the (MRP6) ABCC6 gene generateda stop codon at position 1141 (R1141X; FIG. 1 and Table 2). FIG. 1 cshows the intron/exon structure of the (MRP6) ABCC6 gene. Intron sizesare drawn approximately to scale and the exons are numbered from the 5′end of the (MRP6) ABCC6 gene. FIG. 1 d shows chromatograms of partialDNA sequence from two unrelated PXE patients containing a nonsense and asplice site mutation in exon 24 and intron 21 respectively. In exon 24,the sequence shows a 3421C>T substitution (arrowhead), which wouldgenerate a stop codon at position 1141 (R1141X). PXE patients in aconsanguineous Italian pedigree were found to be homozygous for thisstop codon mutation. In intron 21, a G to T substitution (IVS21+1G>T)was observed within the invariant GT sequence of the donor splice site.This mutation would influence constitutive splicing of (MRP6) ABCC6pre-mRNA and was found in two unrelated PXE patients as a compoundheterozygote in association with either R1141X or R1138Q. FIG. 1 c showsthe sequence of the normal and mutant, nucleotide and amino acidsequences for the nonsense mutation in exon 24 and the splice sitevariant within intron 21.

The C3421T variant in exon 24, which was not found in the control panelof 200 normal alleles, co-segregated in a homozygous form with arecessive PXE phenotype in an Italian family in which all unaffectedindividuals but one were heterozygote carriers (FIG. 4). FIG. 4 shows alarge consanguineous Italian pedigree, SSCP conformers for a homozygousvariant (R1141X) in exon 24 were noted in all four PXE patients (shadedsymbols). All other unaffected family members were heterozygote for thisnonsense mutation except one unaffected family member, indicated by anarrow. SSCP conformers from normal unrelated control DNA have beenincluded. Total RNA from the PXE patient indicated by was used for an RTPCR analysis of (MRP6) ABCC6 mRNA and shown to have low levels of MRP6mRNA.

This R1141X mutation results either in an (MRP6) ABCC6 protein lacking362 amino acids at the C-terminal domain (FIG. 3) or a null allele,produced through nonsense mediated decay of a truncated (MRP6) ABCC6mRNA. Indeed, an analysis of steady state (MRP6) ABCC6 mRNA levels inskin fibroblasts from PXE patients of this Italian pedigree indicatedthe absence of detectable (MRP6) ABCC6 mRNA, suggesting that thehomozygous R1141X mutation results in the total loss of MRP6 geneproduct rather than the production of a truncated protein. R1141X wasalso found in a homozygous state in unrelated patients with autosomalrecessive PXE from the United Kingdom and Belgium. Haplotype analysis ofthe PXE locus in families with the R1141X mutation revealed that thismutation is travelling within different haplotypes, suggesting thatR1141X may be a recurrent mutation.

In two families with a recessive form of PXE from the United Kingdom andthe United States, PXE patients were found to be compound heterozygotes.Affected individuals carried a substitution (TVS21+1G>T) affecting thedonor-splice site of exon 21 of (MRP6) ABCC6, in association with eitherthe nonsense R1141 X substitution in exon 24 or a missense mutation,R1138Q also in exon 24. The splice site mutation occurred at the donorinvariant dinucleotide and lowered the splice potential score from 72.1to 53.8. Several other missense variants (Table 2) were also foundwithin exon 24 and 28 of the (MRP6) ABCC6 gene. These single nucleotidesubstitutions, none of which were detected in the control panel of 200alleles, occurred within highly conserved coding domains, particularlythe domain in exon 28 encoding the Walker A region of the second ATPbinding fold (FIG. 2).

All the detected homozygous or compound heterozygous mutations werefound to be associated with autosomal recessive PXE. One missensemutation (3961G>A) was observed in a family with an apparently dominantform of PXE. All the other heterozygous alterations were detected inindividuals with sporadic PXE. The mode of inheritance of these sporadicPXE cases is presently unknown.

Elastic fibers within elastic tissues such as skin and the arterial wallare fragmented and calcified in PXE patients. Dermal and vascularelastic fiber calcification is patchy and does not involve all elasticfibers in these tissues. Therefore, without wishing to be bound by anyparticular theory, calcification of elastic fibers in PXE is probablytherefore, a secondary consequence of a primary defect of either elasticfiber assembly or the interaction of elastic fibers with otherextracellular matrix components. Accordingly, MRP6 function is morelikely to be related to fiber assembly or matrix interactions thancalcium transport. Another possibility is that the maintenance of theintegrity of normal elastic fibers, extracellular matrix polymerssubject to constant mechanical stress, is modulated by (MRP6) ABCC6 in away that has yet to be explained.

Polymorphic markers in genes encoding known elastic fiber proteins(tropoelastin, lysyl oxidase, fibrillin 1 and 2) were used in a linkageand sib pair analysis, performed with families with both autosomalrecessive (AR) and dominant (AD) forms of PXE. No obvious linkagebetween these markers and the PXE phenotype was found.

TABLE 2 nt change Codon # Effect Location Status UNK gene polymorphisms33C > T 11 Ser to Ser Exon 1 Hetero pM5 gene polymorphisms 2187C > T 729Gly to Gly Exon 19 Hetero 3241G > A 1081 Glu to Lys Exon 28 Hetero MRP-1gene polymorphisms 1062T > C 354 Asn to Asn Exon 9 Hetero 1898G > A 633Arg to Gln Exon 14 Hetero 2001C > T 667 Ser to Ser Exon 16 Hetero2012G > T 671 Gly to Val Exon 16 Both 4002G > A 1334 Ser to Ser Exon 28Hetero IVS29 − 18delT — — Intron 29 Hetero (MRP6) ABCC6 genepolymorphisms 549G > A 183 Leu to Leu Exon 5 Hetero IVS11 − 41A > G — —Intron 11 Both 1841T > C 614 Val to Ala Exon 14 Both 2490C > T 830 Alato Ala Exon 19 Both IVS25 + 90G > A — — Intron 25 Both IVS27 − 46A > G —— Intron 27 Hetero IVS28 + 49C > T — — Intron 28 Hetero 3′UTR + 17G > A— — 3′UTR Hetero (MRP6) ABCC6 gene mutations IVS21 + 1G > T — mRNAIntron 21 Compound splicing 3341G > C 1114 Arg to Pro Exon 24 Homo3413G > A 1138 Arg to Gln Exon 24 Compound 3421C > T 1141 Arg to X Exon24 Compound + Both 3775delT 1259 Fram Shift Exon 27 Hetero 3892G > T1298 Val to Phe Exon 28 Hetero 3904G > A 1302 Gly to Arg Exon 28 Homo3907G > C 1303 Ala to Pro Exon 28 Hetero 3940C > T 1314 Arg toTrp Exon28 Homo 3961G > A 1321 Gly to Ile Exon 28 HeteroTable 2: A summary of all variants identified in the PXE locus in acohort of 20 unrelated PXE patients. Nucleotide (nt) numbering wasderived either from full length published cDNA sequences or fromputative cDNA deduced from genomic DNA sequence. Hetero indicates that avariant was identified in a heterozygous state. Homo indicates that avariant was found in a homozygous state. Both, indicates that a variantwas seen in both heterozygous and homozygous states. Compound, indicatesthat a variant was characterized as a compound heterozygote.

Example 3 Mutation Detection in Dominant Pedigrees

The segregation of A BCC6 mutations with the PXE phenotype was studiedin three pedigrees with an apparent dominant inheritance. Two of thedominant families (families 1 and 3) presented three generations ofindividuals, while the remaining pedigree contained only twogenerations. In all 3 families, a heterozygous mutation, R1141X in exon24, was found to segregate with the PXE phenotype. In the two-generationfamily (family 2), an apparent loss of heterozygosity of the R1141Xallele was detected in the second generation of affected individuals(II-1 to -3). Several polymorphic variants in the surrounding exons andintrons were subsequently analyzed by SSCP. Only one variant, V614A inexon 14, was found to be informative. These results suggested aheterozygous sub-microscopic deletion, which was paternally inherited.This deletion, with a breakpoint between exon 14 and 24, extended beyondexon 24, probably corresponds to a recurrent deletion recentlycharacterized in 4 unrelated families. The latter deletion, confined toa region of the gene between intron 22 and 29 eliminated 16.5 kb ofgenomic DNA. Therefore, individuals II-1, II-2 and II-3 of family 2 haveinherited compound heterozygote mutations, clearly indicating therecessive nature of PXE in this pedigree. Moreover, the phenotypedisplayed by the mother (Individual I-2) carrying a heterozygous alleleR1141X, suggested the partial expression of the phenotype in an obligatecarrier. Indeed, individual I-2 showed discreet skin lesions on the neckassociated with a positive von Kossa staining of a skin biopsy- (fromlesional skin) indicating the presence of calcium salt precipitatestypical of PXE. No angioid streaks were reported for this obligatecarrier and no cardiovascular examination has been performed yet. In theremaining families (family 1 and 3) no other mutations were found.However, the PXE phenotype of the family members dramatically variedwith the generations, clearly suggesting either pseudo-dominance orpartial penetrance in obligate carriers. In family 1, the paternalgrandmother and the father presented discreet skin lesions on the neckregion associated with a positive von Kossa staining of a skin biopsy(no other manifestation were diagnosed), while both children, althoughvery young, had already visible signs of plaques of coalesced papules onthe neck and angioids streaks following ocular examination. In family 3,the paternal grandfather was severely affected with lax and redundantskin, disciform scaring of the retina (the vision is severely impairedat this stage) in addition to active gastrointestinal bleeding andintermittent claudication. The father was only diagnosed with a positivevon Kossa staining of a skin biopsy while 3 of his children presentedwith the characteristic PXE skin lesions and angioid streaks.

Accordingly, heterozygote carriers of PXE mutations can develop PXErelated phenotypes including sub-clinical manifestations of PXE.According to the invention, the penetrance of PXE lesions associatedwith a single mutant (MRP6) ABCC6 allele is between about 10 to 20% ofall carriers, based on the frequency of described AD PXE cases.Therefore, a pedigree with AR PXE presents sub-clinical manifestationsof PXE in 10 to 20% of the obligate carriers. These carriers will beparents of an affected individual and 25% of the unaffected siblings.

Example 4 PXE Heterozygote Frequencies

Upon screening a small sample of the general population (150 normalindividuals) as part of a control panel to verify whether nucleotidesubstitutions found in the (MRP6) ABCC6 gene from PXE patients wereindeed mutations, two heterozygote mutations were found in unrelatedsubjects. The first of these variants was a founder mutation (R1339C)only present in South African Afrikaners while the second substitutionis a recurrent nonsense mutation (R1141X). R1141X is one of the fourrecurrent mutations that have been identified. These mutations are farmore likely to be found in the general population than privatemutations, which, in principle, can only be found in relatedindividuals. The frequency of heterozygote carriers deduced from thepresence of one recurrent mutation in the relatively small sample of thegeneral population is 0.7%. However, four recurrent mutations have thusfar been identified. Although each of the recurrent mutations is likelyto-have a different frequency, the frequency of carriers can be as highas 2.8%, which is consistent with the commonly accepted prevalence ofheterozygote carriers in the general population (0.6 to 2.5%).

Based upon these frequency of heterozygotes and the predicted penetranceof the PXE phenotype in heterozygote carriers (10-20%), heterozygotecarriers with PXE symptoms are expected at a frequency of about 0.25% ofthe general population. In a cohort of about 3000 individuals between 8and 15 persons presenting cardiovascular, ocular or dermal symptomswould be expected. These numbers provide a basis for a statisticalanalysis of the correlation between single (MRP6) ABCC6mutations andpartial manifestations of PXE. Additional cohorts with clinicallydefined cardiovascular abnormalities such as the 1200 sib-pairs groupfrom the Family Blood Pressure Program with hypertension, or the NHLBIFramingham study from which an appropriate cohort of 2400 to 4500individuals is available, can be used to provide additional statisticalsignificance.

Example 5 Creating a Mouse Knockout

To create a knock-out mouse for ABCC6 a neomycin resistance cassette isintroduced between exons 28 and 29 as shown in FIG. 6. This results inthe destruction of the second ATP binding domain whose Walker A domainis encoded by exon 28 and which is essential for the function of any ABCtransporter.

Based on the cDNA sequence for the mouse ABCC6 gene (SEQ ID NO: 8),primers with restriction sites were designed to amplify genomic DNA andallow cloning into vector pPNT described in Tybulewicz et al., Cell vol.65, 1153-1163, 1991. Specifically, a 2.2 kb DNA fragment from exon 26 toexon 28 is cloned into the unique BamHI of pPNT, and a 2.3 kb DNAfragment containing exon 29 to 30 was cloned in the XhoI site of pPNT.In the resulting construct, the neomycin cassette from the vectorinterrupts the reading frame of ABCC6 in exon 28 after the conservedLysine in the walker A domain.

This construct will be linearized by NotI digestion and transfected intomouse 129 stem cells. The two resistance cassettes provided by thevector (TK and Neo) will allow screening for homologous recombinationand knock out of an ABCC6 locus according to methods known in the art(see, for example, Tybulewicz et al., Cell vol. 65, 1153-1163, 1991).

The deletion construct shown in FIG. 6 will be transfected into E. coliand amounts of DNA sufficient for the targeted mutagenesis process willbe produced. This construct will be inserted into embryonic cell linesand cells that incorporate the construct will be implanted intosurrogate mothers and MRP6 null mice will be obtained according tomethods known in the art.

According to the invention, the production of MRP6 null mice withsymptoms resembling those of human PXE would provide further proof thatmutations at the MRP6 locus are responsible for PXE. However, a moreimportant use for MRP6 null mice, or mice that are carriers of an MRP6deletion (heterozygotes having an allele with the MRP6 deletion and awild-type MRP6 allele) is to provide an animal model to study thedevelopment and progression of PXE, and to provide an animal modeluseful in the development of therapeutic approaches (includingidentifying therapeutic drugs) to treat existing PXE or to prevent orreduce the symptoms of PXE before they develop.

Example 6 Examples of Oligonucleotide Probes and Probes Useful to DetectPXE Associated MRP Mutations

Various probes corresponding to regions of specific mutations in ABCC6are used in standard oligonucleotide hybridization, in oligonucleotidearray or nucleic acid chip assays (see www.brownlab.stanford.edu), andin PCR-based techniques for the detection of PXE. Each of the mutationsshown below are indicative of a mutation in the ABCC6 gene that leads toPXE.

In a preferred embodiment, the probe shown in SEQ ID NO: 10 is used forthe detection of a G to A mutation in Exon 24 of the ABCC6 gene.

-   CAGTGGTCCAGGGCATTCCGA (SEQ ID NO: 10)

In another embodiment, the probe shown in SEQ ID NO: 11 is used for thedetection of a C to T mutation in Exon 24 of the ABCC6 gene.

-   CAGTGGTCCGGGCATTCTGA (SEQ ID NO: 11)

In yet another embodiment of the invention, the probe in SEQ ID NO: 12is used for the detection of a G to C mutation in Exon 24 of the ABCC6gene.

-   GACCGTTGGAGTCAGCCAGCTACTCG (SEQ ID NO 12).

In another embodiment of the invention, the probe in-SEQ ID NO: 13 isused for the detection of a C to G mutation in Exon 24 of the ABCC6gene.

-   GACCCTTGGAGTCAGCCAGCTACTGG (SEQ ID NO: 13)

In another embodiment, the following probes are used for the detectionof specific mutations in Exon 26 of the ABCC6 gene.

In a preferred embodiment of the invention, the probe in SEQ ID NO: 14is used for the detection of a C to T mutation in Exon 26 of the ABCC6gene.

-   GGATGTAGGACTATGCCTGGACGCCC (SEQ ID NO: 14)

In a preferred embodiment of the invention, the probe in SEQ ID NO: 15is used for the detection of a G to C mutation in Exon 26 of the ABCC6gene.

-   GGATGCAGGACTATGCCTGCACGCCC (SEQ ID NO: 15)

In yet another preferred embodiment of the invention, specific mutationsin Exon 27 of the ABCC6 gene are detected using the probes shown below.

In a preferred embodiment of the invention, the probe in SEQ ID NO: 16is used for the detection of a C to A substitution in Exon 27 of theABCC6 gene

-   TGCAGCTAAGCCCCCCTGGC (SEQ ID NO: 16)

The probe sequence in SEQ ID NO: 17 is used for the detection of adeletion in Exon 27 of the ABCC6 gene.

-   TGCAGCTCAGCCCCCCGGC (SEQ ID NO: 17)

In yet another embodiment of the invention, the probe in SEQ ID NO: 18is used for the detection of a G to A mutation in Exon 27 of the ABCC6gene.

-   GCTCCAAGCTCCCTGGAGGC (SEQ ID NO: 18)

Mutations in Exon 28 of the ABCC6 gene in patients are detected usingthe probes shown in SEQ ID NOs. 19, 20, 21, 22, 23, 24 and 25.

In a preferred embodiment of the invention, the probe in SEQ ID. 19 isused for the detection of a C to T mutation in Exon 28 of the ABCC6gene.

-   CTGTGGCTCCAGGAGGCAGCTGAGGGTGGG (SEQ ID NO: 19)

In yet another preferred embodiment of the invention,the probe in SEQ IDNO: 20: is used for the detection of a G to A mutation in Exon 28 of theABCC6 gene.

-   CTGCAGCTCCAGGAGGCAGCTGAGGGTGGG (SEQ ID NO: 20)

Similarly, in a preferred embodiment of the invention, the probe in SEQID NO: 21 is used for the detection of a G to A mutation in a differentregion of Exon 28 of the ABCC6 gene.

-   CTGCGGCTCCAGGAGGCAGCTGAGAGTGGG (SEQ ID NO: 21)

Probes in SEQ ID NOs. 22, 23, 24 and 25 are used for the detection ofadditional specific mutations in Exon 28 of the ABCC6 gene.

GTGGGCATCTTTGGCAGGACCGGGG (SEQ ID NO: 22) GTGGGCATCGTTGGCAGGACTGGGG (SEQID NO: 23) GTGGGCATCTTTGGCAGGACCAGGG (SEQ ID NO: 24)GTGGGCATCTTTGGCAGGACCGGGC (SEQ ID NO: 25)

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

INCORPORATION BY REFERENCE

Each of the patent documents and scientific publications disclosedherein is incorporated by reference into this application in itsentirety.

1. A method for identifying a patient as having an increased risk ofhaving children with PXE, the method comprising: a) interrogating anMRP6 nucleic acid in a patient sample for the presence of an MRP6 alleleknown to be a co-segregator with a PXE phenotype; and b) identifyingsaid patient as having an increased risk of having children with PXE ifthe allele from step a) is detected in said-MRP6 nucleic acid.
 2. Amethod for identifying a patient as having an increased risk ofdeveloping a PXE associated symptom, the method comprising: a)interrogating an MRP6 nucleic acid in a patient sample for the presenceof an MRP6 allele known to be a co-segregator with a PXE phenotype; andb) identifying said patient as having an increased risk of developing aPXE associated symptom if the allele from step a) is detected in saidMRP6 nucleic acid.
 3. The method according to claim 2, wherein said PXEassociated symptom is cardiovascular disease.
 4. The method according toclaim 2, wherein said PXE associated symptom is macular degeneration. 5.A method for testing a patient for the presence of a PXE mutation, themethod comprising the steps of: a) interrogating a patient sample for amutation shown to be associated with PXE, the mutation being in the MRP6gene, and the mutation is selected from the group consisting of: i) atcodon 1114, nucleotide 3341G>C; ii) at codon 1138, nucleotide 3413G>A;iii) at codon 1141, nucleotide 3421C>T; iv) at codon 1259, nucleotide3775delT; v) at codon 1298, nucleotide 3892G>T; vi) at codon 1302,nucleotide 3904G>A; vii) at codon 1303, nucleotide 3907G>C; viii) atcodon 1314, nucleotide 3940C>T; and ix) at codon 1321, nucleotide3961G>A; and b) identifying the patient as having a PXE mutation if themutation from step a) is detected in the MRP6 gene.
 6. A method fordetecting a patient as having an increased risk of developing PXE, themethod comprising: a) interrogating an MRP6 nucleic acid in a patientsample; b) determining an abnormal presence or absence of at least onenucleic acid fragment or sequence in the patient's MRP6 nucleic acidcompared to a normal control; and c) identifying said patient as havingan increased risk of developing PXE based on the determination in stepb).
 7. A method for screening a patient for the presence of a MRP6 genemutation, the method comprising: a) interrogating an MRP6 nucleic acidin a patient sample to determine an MRP6 nucleic acid sequence; and b)identifying said patient as having a MRP6 gene mutation by comparing theMRP6 nucleic acid sequence from step a) to a normal MRP6 nucleic acidsequence.